Cistanche Polysaccharides Inhibit Prostate Cancer Cell Proliferation And Induce Apoptosis Via ROS Generation And Antioxidant Gene Suppression

Abstract:
Objective: To investigate the effects and underlying mechanisms of Cistanche polysaccharides on the proliferation and apoptosis of prostate cancer cells.

Methods:
Human prostate cancer DU145 cells were treated with low, medium, and high concentrations of Cistanche polysaccharides (20, 40, and 80 mg/mL, respectively). Cell proliferation was evaluated using the MTT assay, and apoptosis was assessed by flow cytometry. Intracellular reactive oxygen species (ROS) levels were measured via flow cytometry, while glutathione (GSH) levels were determined using ELISA. The mRNA and protein expression levels of glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase modifier subunit (GCLM), and thioredoxin reductase 1 (TXNRD1) were analyzed by RT-PCR and Western blot, respectively.

Results:
The half-maximal inhibitory concentration (IC₅₀) of Cistanche polysaccharides on DU145 cells was 40.35 mg/mL, thus 40 mg/mL was selected as the medium dose for subsequent experiments. The low, medium, and high doses were defined as 50%, 100%, and 200% of the IC₅₀, respectively.

All treatment groups demonstrated significantly increased apoptosis rates compared to the control group. The high-dose Cistanche group exhibited the greatest inhibition of cell proliferation and the highest apoptosis rate, followed by the medium- and low-dose groups (P < 0.01). ROS levels were significantly elevated, and GSH levels were significantly reduced in all Cistanche-treated groups versus control. Notably, the high-dose group had markedly higher ROS and lower GSH levels than the medium- and low-dose groups (P < 0.05); the medium-dose group showed similar differences compared to the low-dose group (P < 0.05).

Expression levels of GCLC, GCLM, and TXNRD1 (both mRNA and protein) were significantly downregulated in all Cistanche-treated groups compared to control (P < 0.05). The high-dose group showed the most significant suppression of these antioxidant-related genes and proteins, followed by the medium- and low-dose groups (P < 0.05).

Conclusion:
Cistanche polysaccharides inhibit the expression of antioxidant factors GCLC, GCLM, and TXNRD1, thereby reducing GSH synthesis, promoting ROS accumulation, and ultimately inducing apoptosis in prostate cancer cells. These findings suggest that Cistanche may serve as a natural therapeutic agent for prostate cancer via oxidative stress-mediated mechanisms.

Cistanche tubulosa-

High polysaccharides Cistanche Extract

Buy Now

Supportive Service Of Wecistanche-The Contact Us To Get A Wholesale Price
Email:wallence.suen@wecistanche.com
Whatsapp/Tel:+86 15292862950

Background and Methods

Prostate cancer accounts for approximately 7.1% of all male malignancies, with peak incidence occurring after the age of 60. As China's population continues to age, the incidence of this disease is expected to rise further [1]. Studies suggest that the high prevalence of prostate cancer in older men may be related to degenerative changes in the prostate, genetic predisposition, dietary habits, and lifestyle factors [2].

Surgical resection remains a mainstay in prostate cancer treatment, aiming to remove most of the tumor mass. However, it is associated with significant complications, such as urinary incontinence, sexual dysfunction, and most critically, tumor recurrence and metastasis, which are major causes of poor prognosis [3].

In recent years, Traditional Chinese Medicine (TCM) has gained increasing attention for its role in cancer therapy. TCM can help boost immunity, counteract chemotherapy/radiotherapy side effects, and enhance detoxification. Some medicinal herbs also exhibit direct antitumor properties, including inhibition of tumor growth and induction of apoptosis in cancer cells [4].

Among these, Cistanche (Cistanche deserticola) is a traditional tonic herb known for its immunomodulatory, antioxidative, and antitumor effects. Its major bioactive components are polysaccharides and phenylethanoid glycosides. This study investigates the effects and underlying mechanism of Cistanche polysaccharides on proliferation and apoptosis of prostate cancer DU145 cells.

Buy Cistanche With 25 Echinacoside and 9 Acteoside

1. Materials and Methods

1.1 Major Materials and Reagents

DU145 human prostate cancer cells were obtained from Tongpai Biotech (Shanghai, China).

Cistanche polysaccharides (Batch No: 0287181) were purchased from Weikeqi Bio Co., Ltd. (Sichuan, China).

Fetal bovine serum, penicillin/streptomycin, and DMEM were provided by Zeta Life (USA).

MTT assay, Annexin V/PI apoptosis kits, RIPA lysis buffer, BCA protein quantification kits were obtained from Elabscience (Shanghai, China).

Reactive oxygen species (ROS) detection kit and GSH ELISA kits were purchased from BioLab (Shanghai, China).

Trizol reagent, reverse transcription kits, and PCR kits were sourced from NeuroSym (Hangzhou, China).

Antibodies against GCLC, GCLM, TXNRD1, β-actin, and corresponding secondary antibodies were purchased from UnivBio (Shanghai, China).

cistanche deserticola

1.2 Cell Culture and Proliferation Assay

DU145 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a CO₂ incubator. For the MTT assay, logarithmic-phase DU145 cells were seeded into 96-well plates and treated with Cistanche polysaccharide solutions at concentrations of 10, 20, 40, and 80 mg/mL. The control group received only DMEM medium.

After 24 hours of treatment, 20 µL of 5 mg/mL MTT was added to each well and incubated for 4 hours. The supernatant was removed, and 150 µL of DMSO was added. After shaking for 10 minutes, absorbance was measured at 490 nm using a microplate reader. The IC₅₀ was calculated, and 20, 40, and 80 mg/mL were selected as low, medium, and high doses for subsequent experiments. The cell proliferation inhibition rate was calculated as:
Inhibition rate (%) = (OD_control - OD_treated) / OD_control × 100

cistanche coffee

1.3 Apoptosis Assay

Log-phase DU145 cells were treated with different concentrations of Cistanche polysaccharides according to groupings. After fixation and staining, Annexin V-FITC and PI were used to label apoptotic cells. Flow cytometry was performed to detect fluorescence signals, and the percentage of apoptotic cells was recorded.

1.4 Detection of Intracellular ROS and GSH Levels

ROS levels were measured using a ROS fluorescence probe and flow cytometry. GSH levels were quantified using ELISA. A standard curve was prepared using known concentrations of GSH standards, and absorbance was measured to calculate GSH concentrations in samples.

1.5 RT-PCR for mRNA Expression of GCLC, GCLM, TXNRD1

Total RNA was extracted using Trizol, and cDNA was synthesized. PCR was performed using specific primers:

GCLC:

Forward: 5'-GTCGGTGTCAAAATACGGAGTTCCA-3'

Reverse: 5'-CGTGACGAGGACTGCGTGCATGGAT-3'

GCLM:

Forward: 5'-CCGATGACGCCATAAACGTCCGTAGC-3'

Reverse: 5'-GGCTAAGCGTACGTAGTACGCAACGTA-3'

TXNRD1:

Forward: 5'-TGACAGTCCTACCGTACGTAGAATGCC-3'

Reverse: 5'-ATAATGCGTAGGGATGCGCTAGCAATGG-3'

β-actin (internal control):

Forward: 5'-TTAGGCGCGATGCAGTCAGATAAGC-3'

Reverse: 5'-ATCGACGTACGTACGGACGCGATAG-3'

Amplification conditions: 95°C for 60 s, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s. PCR products were analyzed by gel electrophoresis, and relative gene expression was normalized to β-actin.

1.6 Western Blotting for Protein Expression

Total protein was extracted using RIPA buffer, and protein concentration was determined by BCA assay. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated overnight at 4°C with primary antibodies against GCLC, GCLM, TXNRD1, and β-actin. After incubation with HRP-conjugated secondary antibodies, bands were visualized using chemiluminescence and quantified by ImageJ software.