Luteoloside inhibiting the prostate cancer cells growth and promoting the tumor cells autophagy through AKT/mTOR pathway

Prostate cancer (PC) is one of the prevalent tumors in men causing higher mortality. Luteoloside has been discovered for suppressive role into the progression of some cancers. However, the Luteoloside’s regulatory functions regarding PC progression are not well understood. This study is thus designed to investigate the Luteoloside impact on PC progression. In this work, it was demonstrated that the PC cell proliferation was gradually inhibited with the increasing Luteoloside dose (0, 20, 40, 80 µ M). Luteoloside also enhanced the PC cell apoptosis. It promoted the autophagy in PC through increasing microtubule-associated protein light chain (LC) 3II/LC3I levels and decreasing p62 levels. Moreover, the Luteoloside reduced phosphorylated-protein kinase B (p-AKT)/AKT and phosphorylated-mechanistic target of rapamycin (p-mTOR)/mTOR levels, indicating that it retarded the AKT/mTOR pathway. In conclusion, the Luteoloside inhibited PC cells growth and promoted tumor cells autophagy through AKT/mTOR pathway. This discovery suggested the Luteoloside as a potential drug in treating PC.


Introduction
Prostate cancer (PC) is one of the leading causes of cancerrelated deaths and impacts men health in the western world [1]. Previous research indicates that~10% of newly diagnosed patients have metastatic lesions which aggravates PC progression [2,3]. Prognoses of late stage and metastatic PC patients is challenging despite the recent global efforts of identifying novel treatment strategies [1,4]. Therapeutic compounds targeting the cellular pathways are therefore important.
Luteoloside (cynaroside or luteolin 7-glucoside) is a naturally abundant flavonoid of plant kingdom [5]. The Luteoloside content has been utilized as quality control index for many Chinese medicinal materials such as honeysuckle, chrysanthemum, and perilla. Luteoloside shows anti-tumor activity, e.g., it modulates the mitogen-activated protein kinas (MAPK) pathway to suppress the cervical cancer cell proliferation and promotes cell apoptosis [6]. Moreover, it retards the AKT/mTOR pathway to attenuate the cell growth, migration, and invasion in gastric cancer [7]. It enhances G0/G1 arrest and autophagy in the non-small cell lung cancer by affecting the Ros-mediated AKT/mTOR/70 kDa ribosomal protein S6 kinase (p70S6K) pathway [8]. Besides, the Luteoloside reduces cell proliferation and metastasis in hepatocellular carcinoma by inhibiting the nucleotide-binding domain and leucinerich repeat related (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome [9]. However, the Luteoloside's role and its mechanism in PC progression are still ambiguous.
DU145 and PC3 cells are the metastatic PC cell lines and represent "castrate resistant" (androgen independent) PC. Luteoloside targets PC cells that are resistant to androgen deprivation therapy "ADT".
In this work, the regulatory functions and related Luteoloside pathway were investigated regarding the PC progression. This discovery revealed that Luteoloside inhibited the PC cells growth and promoted the tumor cells autophagy through AKT/mTOR pathway. This work may assist in treating PC progression by Luteoloside.

Colony formation assay
DU145 and PC3 cells (1000 cells/well) were placed in 6-well plate for 14 days. They were fixed (4% paraformaldehyde) and stained (0.1% crystal violet). The images were taken with a microscope, and colonies were manually counted.

Flow cytometry
The cell apoptosis was measured with Annexin V-fluoresceine isothiocyanate (FITC) Apoptosis Detection Kit (C1062, Beyotime, Shanghai, China). PC cells were washed with phosphate buffer solution (PBS), fixed by ethanol (70%), and centrifuged. The single cell suspension was prepared as 1 × 10 6 cells/mL concentration. PC cells were stained (Annexin V-FITC and Propidium Iodide (PI)) in dark. The cell apoptosis was examined by the flow cytometry (BD FACS Aria III, BD Biosciences, San Jose, CA, USA).

Western blot
The proteins (isolated from PC cells) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and shifted to polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China). After blocking with skimmed milk, the primary antibodies were added in the membranes and kept for 12 h at 4 • C, followed by adding secondary antibodies (1:2000; ab7090) and incubated for 2 h. Lastly, the bands were analyzed with chemiluminescence detection kit (89880, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The protein bands intensity was measured using ImageJ.

Statistical analysis
The data were presented as mean ± standard deviation (SD). Each experiment was performed in triplicate. The statistical analysis was made using GraphPad Prism Software 9 (Graph-Pad Software, San Diego, CA, USA). Student's t-test (for two groups) or one-way analysis of variance (ANOVA) (for multiple groups) were conducted for the comparisons. Posthoc test was performed by Least Significant Difference (LSD) method. p < 0.05 was set as statistically significant.

Luteoloside inhibiting the cell proliferation in PC
The Luteoloside structure is shown in Fig. 1A. DU145 and PC3 cells viabilities were gradually decreased with the increase in Luteoloside dose (0, 20, 40, 80 µM) (Fig. 1B). The colonies were minimized with the increase in Luteoloside dose (0, 20, 40, 80 µM) as monitored through the colony formation assay (Fig. 1C). The data demonstrated that Luteoloside inhibited the PC cell proliferation.
Abnormal activation of the AKT/mTOR pathway has been tracked in variety of cancers, including PC [20,21]. Resultantly, mTOR is phosphorylated and acts as a protein kinase to affect protein synthesis and regulate cell growth, metabolism, and migration by phosphorylating some downstream signaling proteins [22]. Moreover, apoptosis and autophagy are modulated by the AKT/mTOR pathway [23]. In this study, it was found that Luteoloside decreased p-AKT/AKT and p-mTOR/mTOR levels, indicating that Luteoloside retarded the AKT/mTOR pathway.

Conclusions
In conclusion, this study manifested that Luteoloside inhibited the PC cells growth and promoted tumor cells autophagy through AKT/mTOR pathway (Fig. 5). Some limitations however existed, such as lacking human samples, in vivo experiments, and other progresses (angiogenesis, exosome, and immune response). In future, more experiments can be conducted for PC progression to find other regulatory functions of Luteoloside.

F I G U R E 5. Luteoloside inhibiting the AKT/mTOR pathway to reduce cell viability and promote autophagy.
Luteoloside decreased the p-AKT/AKT and p-mTOR/mTOR levels to inhibit the AKT/mTOR pathway. Luteoloside increased LC3II/LC3I and Beclin 1 levels, and decreased p62 levels to strengthen the autophagy. AKT: protein kinase B; mTOR: mechanistic target of rapamycin; LC3: microtubuleassociated protein light chain 3.

AVA IL AB ILI T Y OF DATA AN D M AT E R I A L S
The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request.

A U TH OR CO NT RI BU TI ONS
HYL and LH-designed the study and carried them out; HYL, XW, AYZL, YMP and WZ-supervised the data collection, analyzed the data, interpreted the data; HYL and LHprepared the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

E THICS APPROVAL AND CONSENT TO PA R TICIPATE
Not applicable.

ACK NOWLEDGMENT
Not applicable.

F UNDING
This research received no external funding.