Diosmetin induces apoptosis and protective autophagy in human gastric cancer HGC-27 cells  via the PI3K/Akt/FoxO1 and MAPK/JNK pathways

doi:10.21203/rs.3.rs-3243082/v1

Download PDF

Research Article

Diosmetin induces apoptosis and protective autophagy in human gastric cancer HGC-27 cells via the PI3K/Akt/FoxO1 and MAPK/JNK pathways

https://doi.org/10.21203/rs.3.rs-3243082/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 05 Oct, 2023

Read the published version in Medical Oncology →

You are reading this latest preprint version

Gastric cancer represents a significant global health concern, necessitating the exploration of novel therapeutic options. Diosmetin, a natural flavonoid derived from citrus and vegetables, has demonstrated promising anti-tumor activity against various tumor cells. However, the potential anticancer effect of diosmetin in gastric cancer and its underlying mechanism have yet to be elucidated. In this study, we aimed to investigate the impact of diosmetin on cell proliferation, migration, cell cycle progression and apoptosis in human gastric cancer HGC-27 cells. Our findings revealed that diosmetin effectively suppressed cell proliferation, induced G2/M phase cell cycle arrest, and triggered cell apoptosis. Mechanistically, diosmetin downregulated the expression of antiapoptotic proteins Bcl-2 and Bcl-xL, while upregulated the level of proapoptotic proteins such as Bax, cleaved PARP and cleaved caspase-3. Additionally, diosmetin inhibited Akt and FoxO1 phosphorylation, while activated the MAPK signaling pathway. Notably, pretreatment of IGF-1, an Akt activator, attenuated the diosmetin-induced apoptosis. Furthermore, pretreatment with SP600125, a JNK inhibitor, significantly reduced the protein level of LC3B, while promoted the expression of cleaved caspase-3 and cleaved PARP. Collectively, our results suggest that diosmetin holds promise as an effective therapeutic agent against gastric cancer by inducing apoptosis through inhibition of the Akt/FoxO1 pathway and promoting protective autophagy via the MAPK/JNK signaling pathway.

Gastric cancer

diosmetin

apoptosis

Akt/FoxO1

MAPK/JNK

autophagy

Gastric cancer (GC) is a common form of malignant tumor in gastrointestinal tract cancer. In recent years, gastric cancer is the fifth most common cancer and the third most common cause of cancer death in the world [1]. The development of gastric cancer is related to various risk factors, including Helicobacter pylori (H. pylori) infection, dietary factors, and obesity [2]. Chemotherapy is the established first-line treatment for advanced GC in patients with a good performance status. Studies have shown that chemotherapy can significantly palliate symptoms and improve survival [3]. However, the adverse effects and drug resistance of chemotherapy drugs often lead to suboptimal treatment outcomes and even chemotherapy failure. Therefore, the exploration of novel drugs that offer enhanced efficacy and lower toxicity is crucial.

Apoptosis is a programmed cell death that is regulated by genes, which activates the evolutionarily conserved intracellular pathways to inhibit cancer growth and proliferation [4]. It is primarily mediated by two major pathways: the death receptor pathway and the mitochondrial pathway [5]. Targeting apoptosis has become a prominent strategy in cancer treatment. Autophagy is a fundamental cellular degradation mechanism that involves the breakdown of unnecessary or dysfunctional cellular components through lysosomal machinery [6]. In certain circumstances, tumor cells can survive after chemotherapy due to autophagy by recycling damaged proteins and cellular components to support their growth [7]. Therefore, inhibition of autophagy represents a potential therapeutic strategy against cancer.

Flavonoids have been extensively studied for their anti-tumor properties, showing efficacy against multiple genes and tumor sites with low toxicity [8, 9]. It has been reported that diosmetin induces cell cycle arrest and apoptosis through the activation of p53 pathway [10]. Furthermore, diosmetin has been shown to promote apoptosis through the STAT3/c-Myc signaling pathway in human osteosarcoma cells [11]. However, the specific role of diosmetin in gastric cancer has yet to be elucidated. To gain deeper insights into these effects, we conducted an investigation to explore the impact of diosmetin on gastric cancer cells and elucidate its potential mechanisms of action.

Cell culture

Human Gastric cancer cell line, HGC-27 was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). HGC-27 cells were cultivated in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin–streptomycin. The cells were routinely maintained in a humidified incubator at 37°C with 5% CO₂.

MTS Assay

The MTS assay was performed to quantify cytotoxicity. The cells were then seeded into 96-well plates and exposed to various concentrations of diosmetin (0, 2.5, 5, 10 and 20 µg/mL) for different time intervals (12, 24 and 48 h) in a controlled incubator. Subsequently, 10 µl of Cell Titer 96® Aqueous One Solution Reagent was added each well and incubated for 1 h at 37℃. The plates were measured immediately at 490 nm.

Colony formation assay

A total of 1×10³ cells were seeded in 6-well plates. Subsequently, different concentrations of diosmetin were added, and the cells were further cultured for 7 days. After the incubation period, the colonies were then fixed with 4% paraformaldehyde and stained with crystal violet for 20 min.

Wound‑healing assay

Prior to seeding, equidistant horizontal lines were drawn on the bottom of 6-well plates. Once the cell density reached 90%, a sterile pipette tip was utilized to create perpendicular scratches along the marked lines. Serum-free medium and various concentrations of diosmetin were added to the plates and the cells were cultured for 48 h.

Transwell migration assay

A total of 1.5× 10⁴ cells were seeded in the upper chamber of Transwell insert containing serum-free medium mixed with various concentrations of diosmetin. Subsequently, 600 µL of RPMI-1640 with 10% FBS was added to the lower chamber, the cells in the upper chamber were carefully wiped and then fixed with 4% paraformaldehyde and stained with 2% crystal violet. Finally, the cells were then observed and photographed under a microscope.

Cell cycle analysis

HGC-27 cells were treated with diosmetin for 48 h. Afterwards, they were collected, washed with pre-cooled PBS and then fixed with 70% ethanol overnight. Then the cells were washed again with pre-cooled PBS. Finally, cells were stained with propidium iodide (PI) in the dark at 37°C for 30 min.

Mitochondrial membrane potential analysis using JC-1

HGC-27 cells were treated with diosmetin at different concentrations for 48 h. Subsequently, the cells were collected in centrifugal tube. The cells were then incubated with 5 µl JC-1 fluorescence dye at 37°C for 30 min.

Immunofluorescence Microscopy

HGC-27 cells were seeded onto 12-well tissue culture plates. The cells were treated with different concentrations of diosmetin, with or without Akt activator IGF-1, for 48 h and then fixed with 4% formaldehyde. Following five times washes with PBS containing 0.5% Triton X-100 (PBS-T) for 10 min each, the cells were incubated with primary antibodies (α-Tubulin, cleaved caspase-3) at room temperature for 2 h. The cells were subsequently incubated with the appropriate fluorescently labeled secondary antibodies at room temperature for 1 h. The coverslips were mounted onto microscopy slides using a mounting medium containing DAPI.

TUNEL assay

The cells were treated with varying concentrations of diosmetin with or without Akt activator IGF-1 for 48 h. Cell apoptosis was assessed using the Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). Nuclei were conterstained with Hoechst 33342, and confocal microscopy (Olympus) was utilized to capture images.

Apoptosis Detection by Annexin V- FITC/PI

The cells were plated into a six-well plate and treated with diosmetin for 48 h. All cells in each group were collected, washed twice with PBS. They were then incubated with 5 µL of Annexin V-FITC and 5 µL of PI for 15 min at room temperature in the dark.

Western Blotting

Proteins were extracted using RIPA buffer and denatured protein samples (20–50 µg) were fractionated via SDS-PAGE and transferred to PVDF membranes. The membrane was incubated with primary and secondary antibodies and then incubated with Immobilon Western Chemiluminescent HRP Substrate (Jackson Immuno Research Laboratories) and the bands were visualized under the transillumination of UV light using Azure Biosystems C600.

Statistical Analysis

All experiments were conducted independently and repeated in triplicate. The data are presented as mean ± standard deviation (SD). Statistical analysis was performed using unpaired, two-tailed Student’s t-tests with Bonferroni adjustment or one-way ANOVA for multiple comparisons. A P-value of less than 0.05 was considered statistically significant.

Diosmetin inhibited the proliferation and migration of HGC-27 cells

The chemical structure of diosmetin is depicted in Fig. 1A. To investigate diosmetin’s impact on cytotoxicity in gastric cancer cells, an MTS assay was performed to determine the cell inhibition rate of HGC-27 cells treated with varying concentrations of diosmetin for 48 h. The results demonstrated a clear dose-dependent cytotoxicity of diosmetin (Fig. 1B). As shown in the Fig. 1C, with the increase of diosmetin concentration, fewer colonies were observed compared to the control group. Analyzing the colony numbers further confirmed the dose-dependent inhibitory effect of diosmetin on HGC-27 cell proliferation. Furthermore, we investigated the impact of diosmetin on the migration ability of HGC-27 cells. The area of the wounds was measured at 0 h and at 48 h after diosmetin treatment and migration rates were calculated accordingly. The diosmetin-treated groups showed a significant reduction in cell mobility (Fig. 1D). Additionally, we counted the number of cells that passed through the porous polycarbonate membranes in the cell motility assay. Figure 1E revealed that fewer cells passed through the membranes of the Transwell chambers after being treated with diosmetin. Collectively, these findings indicate that diosmetin exerts a dose-dependent inhibitory effect on both the proliferation and migration of HGC-27 cells.

Diosmetin induced G2/M cell cycle arrest and apoptosis in HGC-27 cells

To investigate the potential correlation between diosmetin-induced cell cycle disruption and its inhibitory effects on the proliferation and migration of HGC-27 cells, we conducted following study. HGC-27 cells were treated with different concentrations of diosmetin for 48 h and then subjected to flow cytometric analysis. The results revealed a notable increase in the percentage of cells in the G2/M fraction as the concentration of diosmetin increased, compared to the untreated control group (Fig. 2A). These findings provide compelling evidence that diosmetin possesses the ability to induce G2/M cell cycle arrest in HGC-27 cells. To further substantiate the potential antitumor mechanism of diosmetin, we proceeded to evaluate its capability to induce apoptosis. The extent of apoptosis in HGC-27 cells provoked by diosmetin was assessed through Annexin V and PI staining, followed by flow cytometric analysis. Following a 48-hour treatment with diosmetin, there was a substantial and concentration-dependent increase in the apoptosis rate. The gradual increase in the proportion of apoptotic cells of HGC-27 cells was illustrated in Fig. 2B. Furthermore, Western blotting analysis revealed that diosmetin dose-dependently increased the levels of cleaved PARP and cleaved caspase-3 proteins, while the levels of full-length caspase-9 and full-length PARP decreased notably (Fig. 2C). Additionally, HGC-27 cells were subjected to staining with α-Tubulin, cleaved caspase-3, and the nuclear stain DAPI, followed by observation under a confocal microscope. Comparing to the control group, the nuclear morphology of diosmetin-treated HGC-27 cells exhibited significant alterations, characterized by increased concentration and even fragmentation. Moreover, the arrangement of α-Tubulin appeared disordered and even disrupted. Notably, the control group exhibited no detectable cleaved caspase-3 expression, whereas the diosmetin treatment group displayed a significant increase (Fig. 2D). These findings strongly suggest that diosmetin treatment induces apoptosis in HGC-27 cells and disrupting the intracellular microtubule network.

Diosmetin induced apoptosis via modulating the Bcl-2 family and disrupting mitochondrial function

The Bcl-2 family of proteins plays a crucial role in regulating cell death by modulating mitochondrial outer membrane permeabilization (MOMP) [12]. We conducted Western blot analysis to assess the changes in protein levels of Bcl-2 family members. Upon exposure to diosmetin, HGC-27 cells exhibited a significant increase in the levels of pro-apoptotic proteins such as p-Bcl2, Bak, and Bax, while the levels of anti-apoptotic proteins including Bcl-2, Bcl-xL, and Bid were dose-dependently down-regulated (Fig. 3A). Flow cytometry analysis showed that diosmetin dose-dependently increased the green to red fluorescence ratio, indicating mitochondrial dysfunction with reduced Δψm (Fig. 3B). These findings strongly support the notion that diosmetin induces apoptosis in HGC-27 cells through the regulation of Bcl-2 family proteins, accompanied by mitochondrial dysfunction.

Diosmetin induced apoptosis by suppressing PI3K/Akt/FoxO1 pathway

Previous studies have emphasized the critical role of the PI3K/Akt/FoxO1 pathway in apoptosis regulation, making it a promising target for cancer therapy [13–15]. Treatment of HGC-27 cells with increasing concentrations of diosmetin resulted in a dose-dependent reduction in the levels of phosphorylated PI3K, Akt, and FoxO1 proteins (Fig. 4A). To further explore the relationship between PI3K/Akt/FoxO1 and apoptosis, we conducted an experiment involving pretreatment of HGC-27 cells with IGF-1, an Akt activator, for 4 hours prior to treatment with 10 µg/mL. Western blotting analysis revealed that pretreatment with IGF-1 effectively reversed the diosmetin-induced decrease in phosphorylation levels of Akt and FoxO1. Additionally, IGF-1 pretreatment significantly attenuated the expression of diosmetin-induced pro-apoptotic proteins, including cleaved PARP, cleaved caspase-3, p-Bcl2, Bak, and Bax (Fig. 4B). Confocal microscopy further demonstrated that pretreatment with IGF-1 reduced the number of cells positive for cleaved caspase-3 in HGC-27 cells treated with diosmetin (Fig. 4C). Moreover, TUNEL staining revealed a significant reduction in HGC-27 cell apoptosis when the cells were pretreated with IGF-1 prior to diosmetin treatment (Fig. 4D). These findings strongly suggest the involvement of the PI3K/Akt/FoxO1pathway in diosmetin-induced apoptosis of HGC-27 cells.

Inhibition of autophagy enhanced diosmetin-induced apoptosis in HGC-27 cells

To assess the impact of diosmetin on autophagy, we examined the levels of LC3B and p62, well-established markers of autophagy. Western blotting analysis revealed that diosmetin treatment significantly increased the level of LC3B, while decreased expression level of p62 compared with the untreated control group (Fig. 5A). To further investigate the relationship between autophagy and apoptosis induced by diosmetin, HGC-27 cells were treated with diosmetin for 48 hours with or without pretreatment with 10 µM chloroquine for 4 h. The western blotting results revealed that chloroquine pretreatment resulted in upregulation in the level of cleaved caspase-3 (Fig. 5B). Furthermore, the combination of diosmetin and chloroquine significantly increased the proportion of apoptotic cells compared to either agent alone (Fig. 5C). These findings provide compelling evidence that autophagy inhibitors effectively enhance diosmetin-induced apoptosis in HGC-27 cells.

MAPK/JNK pathway involved in diosmetin-induced autophagy in HGC-27 cells

The role of MAPK signaling in the regulation of autophagy in cancer cells has been established in previous studies [16]. To investigate the involvement of MAPK signaling in diosmetin-induced autophagy, the phosphorylation levels of MAPK proteins were evaluated. As depicted in Fig. 6A, treatment with diosmetin resulted in a significant and dose-dependent increase in the phosphorylation levels of ERK1/2, p38, and JNK in HGC-27 cells. To provide direct evidence of the relationship between JNK signaling and autophagy, HGC-27 cells were pre-incubated with the JNK inhibitor SP600125 before exposure to diosmetin. The results demonstrated that pretreatment with SP600125 significantly reduced the expression level of the autophagy protein LC3B and concurrently increased the levels of cleaved PARP and cleaved caspase3 proteins in diosmetin-treated HGC-27 cells (Fig. 6B). These findings strongly suggest that diosmetin-induced autophagy and apoptosis is mediated through the activation of the MAPK/JNK pathway.

Cancer is a complex and heterogeneous disease characterized by uncontrolled cell proliferation and disruptions in the cell cycle, leading to the growth and spread of abnormal cells throughout the body [17–19]. Diosmetin, a natural flavonoid with diverse biological activities, has been found to be safe for human use based on acute toxicity studies [20]. Previous research has demonstrated the anti-cancer effects of diosmetin in liver cancer, prostate cancer, and breast cancer, involving mechanisms such as apoptosis promotion and cell cycle arrest [21–23]. In this study, we investigated the potential anti-tumor effect of diosmetin on gastric cancer and explored the underlying mechanisms. Our findings revealed that diosmetin effectively suppressed the proliferation of HGC-27 cells and induced cell cycle arrest, particularly in the G2/M phase. Furthermore, diosmetin triggered mitochondrial apoptosis by inhibiting the PI3K/Akt/FoxO1 pathway and activated protective autophagy through the MAPK/JNK pathway.

Cell cycle arrest and apoptosis are essential targets for effective anti-tumor therapies. Dysregulated cell cycle progression is a major contributor to tumorigenesis, making cell cycle inhibition a critical strategy in cancer treatment [24]. Consistent with previous studies in liver and colorectal cancers, our findings demonstrate that diosmetin induces G2/M cell cycle arrest in HGC-27 cells [21, 25]. The mitochondria play a central role in apoptosis, with Bcl-2 family proteins regulating their permeability[26]. In our study, diosmetin treatment resulted in mitochondrial membrane depolarization, upregulation of pro-apoptotic Bcl-2 family proteins (Bax and Bak), and downregulation of pro-survival proteins (Bcl-2 and Bcl-xL) in HGC-27 cells. Furthermore, increased levels of cleaved caspase-3 and cleaved PARP, recognized markers of apoptosis, were observed, highlighting the activation of the intrinsic pathway by diosmetin-induced apoptosis.

The PI3K/Akt/FoxO1 pathway has emerged as a promising target for cancer therapy due to its involvement in apoptosis regulation [27]. In our study, we observed a significant inhibition of the PI3K/Akt/FoxO1 signaling pathway by diosmetin. Interestingly, pretreatment with the Akt activator IGF-1 resulted in a substantial increase in the levels of phosphorylated Akt, and FoxO1 proteins in diosmetin-treated HGC-27 cells. Strikingly, this IGF-1 pretreatment also led to a notable decrease in the expression levels of key pro-apoptotic proteins, including cleaved caspase-3, cleaved PARP, Bax, Bak, and p-Bcl2. These observations align with previous reports that have demonstrated a correlation between high expression of p-FoxO1 and poor prognosis in gastric cancer [28]. Notably, our findings are consistent with the study by Wu et al., which reported that the phosphorylation-mediated inactivation of the PI3K/Akt/FoxO1 pathway suppresses proliferation and regulates the cell cycle in prostate cells [29]. Based on these collective pieces of evidence, we conclude that diosmetin induces mitochondrial apoptosis in HGC-27 cells by inactivating the PI3K/Akt/FoxO1 signaling pathway.

In our study, we aimed to investigate the interplay between JNK, autophagy, and apoptosis in our experimental model. We pretreated HGC-27 cells with the JNK inhibitor SP600125. The inhibition of JNK resulted in decreased levels of the autophagy marker protein LC3B, along with increased levels of apoptotic proteins, including cleaved caspase-3 and cleaved PARP, in diosmetin-treated cells. These findings align with previous studies suggesting that JNK positively regulates autophagy and contributes to cell survival [30]. Notably, JNK1 has been implicated in promoting cell survival in gastric cancer [31], further supporting our results. The dual role of JNK in regulating both autophagy and apoptosis highlights its complex involvement in cellular processes and underscores the need for further investigations to elucidate the precise molecular mechanisms underlying this interplay.

Our study represents the first investigation into the antitumor effects of diosmetin on human gastric cancer cells. Diosmetin exerts its apoptotic effects through multiple pathways (Fig. 6C). Firstly, it inhibits the PI3K/Akt/FoxO1 signaling pathway, resulting in the regulation of Bcl-2 family proteins and mitochondrial dysfunction, ultimately triggering apoptosis. Secondly, diosmetin activates the MAPK/JNK signaling pathway, which induces protective autophagy. Together, these processes contribute to the overall apoptotic response mediated by diosmetin in gastric cancer cells. These results underscore the potential of diosmetin as a promising therapeutic agent for the treatment of gastric cancer. Moreover, the combination of diosmetin with an autophagy inhibitor may further enhance its efficacy.

HGC-27	Human gastric cancer cell line
Bcl-2	B-cell lymphoma 2
Bcl-xL	B-cell lymphoma-extra large
Bax	Bcl-2-associated X protein
PARP	Poly (ADP-ribose) polymerases
Akt	Protein kinase B
FoxO1	Forkhead box protein O1
MAPK	Mitogen-activated protein kinase
JNK	Jun N-terminal kinase
LC3B	Microtubule Associated Protein 1 Light Chain 3 Beta
GC	Gastric cancer
MTS	3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
FBS	Fetal bovine serum
PI	propidium iodide
JC-1	5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine, iodide
DAPI	4′,6-diamidino-2-phenylindole
TUNEL	Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling

Acknowledgements This research was supported by the Competitive Allocation Project of Zhanjiang Municipal Science and Technology Development Special Fund (2021A05124 and 2022A01165).

Conflict of interest The authors declare that they have no potential conflicts of interest.

Ethical approval Not applicable.

Consent to participate Not Applicable.

Consent for publication All authors consent to the publication of this.

Smyth EC, Nilsson M, Grabsch HI, van Grieken NC, Lordick F. Gastric cancer: Lancet. 2020;396(10251):635–48.
Thrift AP, Wenker TN, El-Serag HB. Global burden of gastric cancer: epidemiological trends, risk factors, screening and prevention. Nat Rev Clin Oncol. 2023;20(5):338–49.
de Jongh C, van der Veen A, Brosens LAA, Nieuwenhuijzen GAP, Stoot J, Ruurda JP, van Hillegersberg R, Group LS. Distal Versus Total D2-Gastrectomy for Gastric Cancer: a Secondary Analysis of Surgical and Oncological Outcomes Including Quality of Life in the Multicenter Randomized LOGICA-Trial: J Gastrointest Surg. 2023.
Xu Q, Pan G, Wang Z, Wang L, Tang Y, Dong J, Qin JJ. Platycodin-D exerts its anti-cancer effect by promoting c-Myc protein ubiquitination and degradation in gastric cancer. Front Pharmacol. 2023;14:1138658.
Lossi L. The concept of intrinsic versus extrinsic apoptosis. Biochem J. 2022;479(3):357–84.
Lin NY, Beyer C, Giessl A, Kireva T, Scholtysek C, Uderhardt S, Munoz LE, Dees C, Distler A, Wirtz S, et al. Autophagy regulates TNFalpha-mediated joint destruction in experimental arthritis. Ann Rheum Dis. 2013;72(5):761–8.
Lim SM, Mohamad Hanif EA, Chin SF. Is targeting autophagy mechanism in cancer a good approach? The possible double-edge sword effect. Cell Biosci. 2021;11(1):56.
Dai QS, Liu W, Wang XB, Lu N, Gong DD, Kong LY, Guo QL. NCPMF-60 induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Anticancer Drugs. 2011;22(1):46–57.
Hu B, Shen KP, An HM, Wu Y, Du Q. Aqueous extract of Curcuma aromatica induces apoptosis and G2/M arrest in human colon carcinoma LS-174-T cells independent of p53. Cancer Biother Radiopharm. 2011;26(1):97–104.
Liu B, Zhang M, Liu S, Ying J, Zhang J, Kurihara H, Zheng W, He RR, Zhu R. Diosmetin, a Potential p53 Activator, Performs Anticancer Effect by Regulating Cell Cycling and Cell Proliferation in HepG2 Cells. Protein Pept Lett. 2017;24(5):413–8.
Ning R, Chen G, Fang R, Zhang Y, Zhao W, Qian F. Diosmetin inhibits cell proliferation and promotes apoptosis through STAT3/c-Myc signaling pathway in human osteosarcoma cells. Biol Res. 2021;54(1):40.
Kale J, Osterlund EJ, Andrews DW. BCL-2 family proteins: changing partners in the dance towards death. Cell Death Differ. 2018;25(1):65–80.
Zhang N, Qiu J, Zheng T, Zhang X, Hua K, Zhang Y. Goserelin promotes the apoptosis of epithelial ovarian cancer cells by upregulating forkhead box O1 through the PI3K/AKT signaling pathway. Oncol Rep. 2018;39(3):1034–42.
Cui H, Zhang Q. Dexmedetomidine ameliorates lipopolysaccharide-induced acute lung injury by inhibiting the PI3K/Akt/FoxO1 signaling pathway. J Anesth. 2021;35(3):394–404.
Li W, Cheng M, Zhang W, He R, Yang H. New Insights into the Mechanisms of Polyphenol from Plum Fruit Inducing Apoptosis in Human Lung Cancer A549 Cells Via PI3K/AKT/FOXO1 Pathway. Plant Foods Hum Nutr. 2021;76(1):125–32.
Shen F, Ge C, Yuan P. Aloe-emodin induces autophagy and apoptotic cell death in non-small cell lung cancer cells via Akt/mTOR and MAPK signaling. Eur J Pharmacol. 2020;886:173550.
Neagu M, Constantin C, Popescu ID, Zipeto D, Tzanakakis G, Nikitovic D, Fenga C, Stratakis CA, Spandidos DA, Tsatsakis AM. Inflammation and Metabolism in Cancer Cell-Mitochondria Key Player. Front Oncol. 2019;9:348.
Kroemer G, Pouyssegur J. Tumor cell metabolism: cancer's Achilles' heel. Cancer Cell. 2008;13(6):472–82.
Korgaonkar N, Yadav KS. Understanding the biology and advent of physics of cancer with perspicacity in current treatment therapy. Life Sci. 2019;239:117060.
Adamante G, de Almeida AS, Rigo FK, da Silva Silveira E, Coelho YO, De Pra SD, Milioli AM, Camponogara C, Casoti R, Bellinaso F, et al. Diosmetin as a novel transient receptor potential vanilloid 1 antagonist with antinociceptive activity in mice. Life Sci. 2019;216:215–26.
Ma A, Zhang R. Diosmetin Inhibits Cell Proliferation, Induces Cell Apoptosis and Cell Cycle Arrest in Liver Cancer. Cancer Manag Res. 2020;12:3537–46.
Oak C, Khalifa AO, Isali I, Bhaskaran N, Walker E, Shukla S. Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest. Int J Oncol. 2018;53(2):835–43.
Wang C, Li S, Ren H, Sheng Y, Wang T, Li M, Zhou Q, He H, Liu C. Anti-Proliferation and Pro-Apoptotic Effects of Diosmetin via Modulating Cell Cycle Arrest and Mitochondria-Mediated Intrinsic Apoptotic Pathway in MDA-MB-231 Cells. Med Sci Monit. 2019;25:4639–47.
Liu J, Peng Y, Wei W. Cell cycle on the crossroad of tumorigenesis and cancer therapy. Trends Cell Biol. 2022;32(1):30–44.
Koosha S, Mohamed Z, Sinniah A, Alshawsh MA. Investigation into the Molecular Mechanisms underlying the Anti-proliferative and Anti-tumorigenesis activities of Diosmetin against HCT-116 Human Colorectal Cancer. Sci Rep. 2019;9(1):5148.
Sarkar A, Paul A, Banerjee T, Maji A, Saha S, Bishayee A, Maity TK. Therapeutic advancements in targeting BCL-2 family proteins by epigenetic regulators, natural, and synthetic agents in cancer. Eur J Pharmacol. 2023;944:175588.
Li F, Chen L, Zheng J, Yang J, Song X, Wang Y, Zhou X. Mechanism of Gegen Qinlian Decoction Regulating ABTB1 Expression in Colorectal Cancer Metastasis Based on PI3K/AKT/FOXO1 Pathway. Biomed Res Int. 2022;2022:8131531.
Altan B, Yokobori T, Ide M, Mochiki E, Toyomasu Y, Kogure N, Kimura A, Hara K, Bai T, Bao P, et al. Nuclear PRMT1 expression is associated with poor prognosis and chemosensitivity in gastric cancer patients. Gastric Cancer. 2016;19(3):789–97.
Wu Z, Sun H, Zeng W, He J, Mao X. Upregulation of MircoRNA-370 induces proliferation in human prostate cancer cells by downregulating the transcription factor FOXO1. PLoS ONE. 2012;7(9):e45825.
Almasi S, Kennedy BE, El-Aghil M, Sterea AM, Gujar S, Partida-Sanchez S, El Hiani Y. TRPM2 channel-mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway. J Biol Chem. 2018;293(10):3637–50.
Tripathi D, Cheekatla SS, Paidipally P, Radhakrishnan RK, Welch E, Thandi RS, Tvinnereim AR, Vankayalapati R. c-Jun N-terminal kinase 1 defective CD4⁺CD25⁺FoxP3⁺ cells prolong islet allograft survival in diabetic mice. Sci Rep. 2018;8(1):3310.

Download PDF

Journal Publication

published 05 Oct, 2023

Read the published version in Medical Oncology →

Editorial decision: Accept as is
01 Sep, 2023
Reviewers agreed at journal
20 Aug, 2023
Reviewers invited by journal
20 Aug, 2023
Editor assigned by journal
09 Aug, 2023
First submitted to journal
08 Aug, 2023

You are reading this latest preprint version

Diosmetin induces apoptosis and protective autophagy in human gastric cancer HGC-27 cells via the PI3K/Akt/FoxO1 and MAPK/JNK pathways

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

Cell culture

MTS Assay

Colony formation assay

Wound‑healing assay

Transwell migration assay

Cell cycle analysis

Mitochondrial membrane potential analysis using JC-1

Immunofluorescence Microscopy

TUNEL assay

Apoptosis Detection by Annexin V- FITC/PI

Western Blotting

Statistical Analysis

Results

Diosmetin inhibited the proliferation and migration of HGC-27 cells

Diosmetin induced G2/M cell cycle arrest and apoptosis in HGC-27 cells

Diosmetin induced apoptosis via modulating the Bcl-2 family and disrupting mitochondrial function

Diosmetin induced apoptosis by suppressing PI3K/Akt/FoxO1 pathway

Inhibition of autophagy enhanced diosmetin-induced apoptosis in HGC-27 cells

MAPK/JNK pathway involved in diosmetin-induced autophagy in HGC-27 cells

Discussion

Conclusion

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1