The Tanshinones (Tan) Extract From Salvia miltiorrhiza Bunge Induces ROS-Dependent Apoptosis in Pancreatic Cancer via AKT Hyperactivation-Mediated FOXO3/SOD2 Signaling

Abstract

Context:

Salvia miltiorrhiza (SM) is a commonly used herb in traditional Chinese medicine (TCM) and has been used in the treatment of pancreatic cancer to relieve the symptom of “blood stasis and toxin accumulation.” Tanshinones (Tan), the main lipophilic constituents extracted from the roots and rhizomes of SM, have been reported to possess anticancer functions in several cancers. But the mechanism of how the active components work in pancreatic cancer still need to be clarified.

Objective:

In this study, we aimed to investigate the therapeutic potential of Tan in pancreatic cancer and elucidate the underlying mechanisms.

Materials and Methods:

The viabilities of PANC-1 and Bxpc-3 cells were determined by MTT assay, after treatment with various concentrations of Tan. The apoptotic cells were quantified by annexin V-FITC/PI staining and DAPI staining assays. The expression of relative proteins was used western blotting. Tumor growth was assessed by subcutaneously inoculating cells into C57BL/6 mice.

Results:

Our experiments discovered that Tan effectively suppressed pancreatic cancer cell proliferation and promoted apoptosis. Mechanistically, we propose that Tan enhances intracellular ROS levels by activating the AKT/FOXO3/SOD2 signaling pathway, ultimately leading to apoptosis in pancreatic cancer cells. In vivo assay showed the antitumor effect of Tan.

Conclusion:

Tan, a natural compound from Salvia miltiorrhiza, was found to effectively suppress pancreatic cancer cell proliferation and promote apoptosis both in vitro and in vivo. Mechanistically, we propose a positive feedback loop mechanism. These findings provide valuable insights into the molecular pathways driving pancreatic cancer progression.

Graphical Abstract

Mechanisms of Tan-induced apoptosis in pancreatic cancer cells Tan enhances intracellular ROS levels by activating the AKT/FOXO3/SOD2 signaling pathway, ultimately leading to apoptosis in pancreatic cancer cells.

Introduction

Pancreatic cancer (PC) is a highly aggressive and malignant tumor with limited treatment options and a poor prognosis, with a 5-year survival rate of less than 9%.^1,2 Currently, only a small percentage of PC patients (10%-20%) qualify for curative surgery due to late diagnosis. ³ Chemotherapy, such as gemcitabine and albumin-bound paclitaxel, or modified FOLFIRINOX, is the standard first-line treatment for unresectable pancreatic cancer. ⁴ However, the majority of patients with advanced or metastatic disease show poor response to chemotherapy and experience severe adverse reactions. Therefore, exploring natural compounds from traditional medicines as potential agents for pancreatic cancer treatment is a promising approach to improve patient outcomes.

Over the past 3 decades, the mechanism of clinical cancer therapy, involving chemotherapeutic agents and ionizing radiation, has been associated with increased production of Reactive Oxygen Species (ROS), leading to programmed cell death or apoptosis. ⁵ ROS play a role in various cellular processes, including cell proliferation, differentiation, and apoptosis. ⁶ The Nrf2/HO-1 pathway, regulated by ROS, plays a crucial role in maintaining redox balance and reducing oxidative stress. ⁷ ROS can have dual effects in pancreatic cancer, promoting cancer progression at moderate levels but selectively damaging cancer cells at high levels. ⁸ Tumor cells have higher levels of ROS compared to normal cells due to their abnormal metabolism and increased oxidative stress. Recent studies have shown that increasing ROS levels can induce cell death and enhance the efficacy of chemotherapy. In colon cancer, Dendrobium officinale polysaccharide disrupts mitochondrial function by increasing ROS levels, thereby activating the AMPK/mTOR autophagy signaling pathway and inducing cell death. ⁹ Similarly, in pancreatic ductal adenocarcinoma (PDAC), hernandezine demonstrates anticancer effects on Capan-1 and SW1990 cells by activating the AMPK pathway and enhancing ROS generation in pancreatic cancer cells. ¹⁰ Based on these mechanisms, increasing ROS levels has emerged as an important strategy for inducing tumor cell death. Exploring new components that can selectively increase ROS levels in pancreatic cancer cells may offer a promising strategy for cancer treatment.

AKT, a protein kinase, is often hyperactivated in cancer cells, promoting their growth and survival. ¹¹ Targeting AKT has been explored as an anti-cancer therapy, but pan-Akt inhibition can lead to adverse effects such as hyperglycemia and diabetes.^12,13 FOXO3, part of the FOXO family of transcription factors, is regulated by AKT and exhibits both antioxidant and anticancer activities. ¹⁴ When AKT is activated, FOXO is phosphorylated and confined to the cytoplasm, leading to the loss of its transcriptional activity. FOXO3 is generally considered a tumor suppressor, but its role in tumorigenesis is complex and context-dependent. High expression of FOXO3 has been associated with poor survival outcomes in hepatocellular cancer and shorter overall survival in pancreatic cancer. ¹⁵ In pancreatic cancer, patients with high activation signatures of FOXO3 exhibit a shorter overall survival rate compared to those with low activation levels. ¹⁶ FOXO3 also promotes the invasive properties of cancer cells by increasing the expression of matrix metalloproteinases. ¹⁷ Furthermore, FOXO3 plays a crucial role in cellular defense against oxidative stress by regulating ROS scavengers such as SOD2. SOD2 helps reduce intracellular levels of ROS. Various studies have shown that modulating the AKT/FOXO3/SOD2 pathway can enhance the effectiveness of cancer treatment. For example, previous study indicated that the induction of apoptosis in hepatocellular carcinoma cells by Juglanthraquinone C involves the activation of the Akt/FOXO3 signaling pathway and elevation of intracellular ROS levels. ¹⁸ Hyperactivation of AKT leads to inactivation of FOXO3 and then fails to initiate the transcription of SOD2, leading to inadequate clearance of ROS and promoted apoptosis of hepatocellular carcinoma cells. ¹⁹ In pancreatic cancer cells, metformin increased ROS levels dramatically via AMPK-FOXO3a-MnSOD pathway and combination of metformin/apigenin exerts anticancer activity through DNA damage-induced apoptosis, autophagy, and necroptosis. ²⁰ Additionally, PPARγ/SOD2 prevents mitochondrial ROS-induced apoptosis by inhibiting ATG4D-mediated mitophagy and promotes pancreatic cancer cell proliferation. ²¹ Experimental suppression of AKT-dependent phosphorylation of FoxO3a in colon and breast cancer cell lines has been shown to result in transcriptional downregulation of its downstream target genes such as Cu/ZnSOD, MnSOD, and catalase. This molecular disruption causes an increase in the production of reactive oxygen species (ROS), which leads to the induction of premature senescence in the cancer cells. ²² Therefore, targeting this pathway represents a potential strategy for cancer therapy with significant clinical relevance.

Salvia miltiorrhiza (SM) is an herb used in traditional Chinese medicine (TCM) that has gained attention for its pharmacological properties. Tan, the main lipophilic constituents extracted from the roots and rhizomes of SM, have been reported to possess several biological functions, including anti-inflammatory, anticoagulant, anticancer, and antibacterial properties. ²³ In recent years, studies have focused on identifying Tan as apoptosis-inducing compounds for cancer cells. For example, Tan IIA has been found to inhibit colorectal cancer cell growth by activating the ROS/JNK signaling pathway, leading to cell autophagy and apoptosis. ²⁴ Dihydrotanshinone triggers apoptosis in HepG2 cells through the ROS-mediated p38 MAPK signaling pathway. ²⁵ Tanshinone I induces apoptosis in liver cancer cells by triggering ROS-mediated endoplasmic reticulum stress and inhibiting autophagy. ²⁶ These findings suggest the potential of Tan as therapeutic agents for cancer treatment. However, the specific signaling pathways involved in Tan-induced apoptosis in pancreatic cancer cells require further investigation.

In this study, we aimed to investigate the therapeutic potential of Tan for pancreatic cancer and elucidate the underlying mechanisms. We used network pharmacology to identify potential targets of Tan in combating pancreatic cancer. Through molecular docking and similarity analysis, we predicted active components of Tan and identified crucial targets related to these components within pancreatic cancer using a protein-protein interaction (PPI) network. Our experimental results revealed that Tan affects cell proliferation and apoptosis in pancreatic cancer cells through the production of reactive oxygen species (ROS). Specifically, we investigated the AKT/FOXO3/SOD2 pathway, which plays a critical role in regulating ROS and apoptosis. Tan activated AKT, leading to increased phosphorylation and nuclear exclusion of FOXO3. This resulted in reduced expression of SOD2, leading to the accumulation of intracellular ROS and subsequent apoptosis in pancreatic cancer cells. Furthermore, we validated the inhibitory effect of Tan on pancreatic tumor growth in vivo using a mouse model. Western blotting and immunohistochemistry confirmed that Tan inhibits pancreatic cancer tumor growth by activating the AKT/FOXO3/SOD2 pathway. These findings provide important insights into the molecular pathways driving pancreatic cancer progression and suggest the potential of developing targeted therapies using natural compounds like Tan.

Materials and Methods

Cell Culture and Reagents

The human pancreatic cancer cell line BxPC-3 was obtained from the Beijing Beina Chuanglian Biotechnology Research Institute. The PANC-1 cell line was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China. The cells were cultured in RPMI 1640 (Gibco Life Technologies) or DMEM (Gibco Life Technologies) supplemented with 10% FBS (Sijiqing Biotechnology, Zhejiang, China) at 37°C in a humidified atmosphere containing 5% CO₂. The N-acetylcysteine (NAC) and LY294002 reagents were purchased from Beyotime Biotechnology.

Chromatographic Conditions of HPLC

Tanshinones was dissolved in methanol at a concentration of 1.0 mg/mL and subsequently analyzed using the Agilent 1260 Series HPLC system. A Thermo Fisher Dionex Acclaim C18 column (4.6 × 250 mm², 5 µm) was used for the analysis. The mobile phase consisted of a 0.02% formic acid aqueous solution and acetonitrile under isocratic elution conditions. The flow rate was set at 0.8 mL/min. The ELSD drift tube temperature was maintained at a constant 25°C throughout the analysis.

Cell Viability Assay

Cell viability was assessed using MTT assay with reference to previous study reports ²⁷ and according to the protocol provided by the manufacturer (Beyotime Biotechnology, China). PANC-1 and BxPC-3 pancreatic cancer cells were seeded in 96-well plates at a density of 8 × 10³ cells per well in 100 μL of DMEM (Dulbecco’s Modified Eagle Medium) or RPMI 1640 medium supplemented with 10% FBS (fetal bovine serum). The cells were incubated overnight at 37°C in a humidified incubator with 5% CO₂. After the overnight incubation, the medium was replaced with Tan solution, a treatment solution whose composition was not specified, but was diluted in the medium to achieve a final concentration range of 2.5 to 40 μg/mL. The cells were then further incubated for 24 or 48 hours. After the incubation period, cell viability was evaluated by treating each well with a 5 mg/mL MTT solution and incubating the cells for an additional 4 hours. The formazan crystals were dissolved in 150 μL of DMSO, and the absorbance at 490 nm was measured using a microplate reader. The absorbance readings were used to calculate cell viability. The entire experiment was performed in triplicate with 3 independent replicates. Safety precautions, such as wearing gloves and a mask, and conducting the experiment in a biosafety cabinet, were followed during the experimental procedures.

DAPI Staining

A DAPI solution (Beyotime Biotechnology, China) was used to label apoptotic cells. Prior to staining, cell density in an appropriate culture medium (DMEM supplemented with 10% FBS) was adjusted to 5 × 10⁵ cells/mL. After treating the cells with different concentrations of Tan for 48 hours, the culture medium was carefully removed, and the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. The fixed cells were then washed 3 times with PBS before being incubated with the DAPI solution (5 μg/mL) for 15 minutes at room temperature in the dark. Subsequently, the cells underwent 2 additional PBS washes and were examined using fluorescence microscopy (excitation at 360 nm, emission at 460 nm). Apoptotic cells were identified based on characteristic nuclear condensation and fragmentation. The experiment was conducted in triplicate, and a minimum of 200 cells per condition were analyzed.

Flow-Cytometry Analysis

Referring to previous studies,^28,29 Annexin V-FITC/PI double labeling was used to measure apoptosis in pancreatic cancer cells. The cells were incubated at 37°C and 5% carbon dioxide. They were subsequently subjected to 48 hours of Tan treatment at varied doses (2.5-20 mg/mL). After treatment, the cells were harvested and washed twice with PBS for 5 minutes each wash. Subsequently, the suspended cells (cell suspension volume: 100 μL) were supplemented with 5 μL of Annexin V-FITC conjugate and 5 μL of PI in 100 μL of binding buffer. The mixture was incubated for 20 minutes in the dark at an undisclosed temperature. Sequentially, 400 μL of Annexin V-FITC/PI binding buffer was introduced, and the cells were subsequently collected for quantitative analysis using Agilent NovoCyte flow cytometry.

Measurement of ROS

The levels of Reactive Oxygen Species (ROS) were quantified utilizing a Reactive Oxygen Species Assay Kit, obtained from Shanghai Beyotime. The cells were seeded in 6-well plates and treated with various concentrations of Tan for 48 hours. Afterward, the cells were collected and suspended in 200 μL of serum-free medium containing 10 μM DCFH-DA, and incubated at 37°C for 20 minutes. After incubation, the cells were rinsed thrice with serum-free medium prior to collection for quantitative analysis. Flow cytometry was employed using Agilent NovoCyte for the analysis. Intracellular ROS generation was evaluated by measuring fluorescence using flow cytometry. Appropriate positive and negative controls were included in the experiment for validation. Similarly, ROS fluorescence was observed using an inverted fluorescence microscope after cells were treated with the DCFH-DA probe.

Detection of Mitochondrial Membrane Potential (MMP)

MMP was measured by JC-1 method as previous study. ³⁰ Briefly, the cells were seeded on 6-well plates (2 × 10⁵ cells/well) and cultured overnight at 37°C. After discarding the supernatant, the cells were treated with Tan or NAC pretreatment and cultured for 24 hours in a 5% CO₂ incubator at 37°C. Following that, cells were mixed with JC-1 staining working solution, and further cultured at 37°C for 30 minutes, and then washed with PBS for 3 times. Five different fields of vision of the same section were observed by fluorescence microscope. The JC-1 kit was purchased from Beyotime.

RNA Preparation, qRT-PCR, and RNA Sequencing

Total RNA was extracted from PANC-1 using the Total RNA Extraction Kit (Yisheng, China), and cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Yisheng, China), following the manufacturer’s protocol. Quantitative real-time PCR was performed using the SYBR Premix Ex Taq II kit (Yisheng, China). The primer sequences are listed in Supplemental Table 1. Gene expression levels were normalized to Actin, and relative quantification was performed using the 2^−ΔΔCt method.

Western Blot Analysis

The harvested cells treated with Tan were lysed using RIPA buffer (Fdbio Science, China), specifically RIPA lysis buffer containing protease and phosphatase inhibitors at a dilution of 1:10. The concentration of protein was determined using the BCA protein assay kit (Sangon Biotech, Shanghai), following the manufacturer’s instructions. Equal amounts of protein (30 μg) were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a concentration range of 8% to 12%. The proteins were separated by gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% defatted milk in phosphate-buffered saline (PBS) for 2 hours at room temperature. They were then incubated overnight at 4°C with primary antibodies. The primary antibodies used included β-Actin, Cleaved caspase3, Cleaved caspase9, Cleaved PARP, Survivin, Bcl-2, c-Myc, STAT3, p-Stat3, Akt, p-Akt, FOXO3, p-FOXO3, P62 and LC3 II (Cell Signaling Technology, USA), as well as SOD2 (Huabio, Hangzhou). The dilution ratios for primary antibodies were as follows: 1:1000 for β-Actin, Cleaved caspase3, Cleaved caspase9, Cleaved PARP, Survivin, Bcl-2, c-Myc, STAT3, p-Stat3, Akt, p-Akt, and FOXO3; and 1:500 for p-FOXO3 and SOD2. Afterward, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (dilution 1:5000) for an additional 2 hours at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) reagent (Fdbio Science), and the data were normalized to β-Actin.

Immunofluorescence Staining

After completing the designated treatment, the cells were washed 3 times with phosphate-buffered saline (PBS) and fixed with 4% formaldehyde for 15 minutes. Subsequently, the cells were permeabilized using 0.3% Triton X-100 and then blocked with a solution of 5% bovine serum albumin diluted in PBS for 1 hour at room temperature to minimize nonspecific binding. For immunostaining, the cells were incubated overnight at 4°C with primary antibodies against Nrf2 and FOXO3. The primary antibodies used in this study were anti-Nrf2 (dilution ratio: 1:500) and anti-FOXO3 (dilution ratio: 1:200). After the overnight incubation, the slides were washed 3 times with PBS for 10 minutes per wash to remove any unbound primary antibodies. Subsequently, the cells were incubated with a Texas Red-conjugated secondary antibody for 1 hour at room temperature. Finally, the slides were imaged using a fluorescence microscope (Zeiss Instruments confocal microscope) with appropriate excitation and emission spectra at 400× magnification.

Immunohistochemistry

Paraffin-embedded tissue sections were deparaffinized by a series of ethanol baths, followed by rehydration. To block endogenous peroxidase activity, the sections were then incubated in methanol with 0.3% hydrogen peroxide for 10 minutes at room temperature. Antigen retrieval was induced by heating the tissue slides in a pH 6.0 antigen retrieval solution for 30 minutes. Subsequently, overnight incubation of the slides with PCNA and survivin antibodies (Cell Signaling Technology, USA) was conducted at 4°C. Staining was conducted using the Prolink-2 Plus HRP rabbit polymer detection kit (Golden Bridge, Bothell, WA, USA), following the manufacturer’s instructions. Image acquisition was performed using Aperio ScanScope CS software (Aperio Technologies, Vista, CA, USA).

Targets Filter

The chemical constituents of Tan were retrieved from the TCMID database (https://ngdc.cncb.ac.cn/databasecommons/database/id/437). All the chemical constituents were then subjected to the Swisstargets database (http://www.swisstargetprediction.ch/) to screen parameters related to absorption, distribution, metabolism, and excretion (ADME). The compounds that met the required criteria were selected. Additionally, the active targets were searched using the Swisstargets and Batman databases (http://bionet.ncpsb.org/batman-tcm/) based on “pancreatic cancer” as the keyword. Disease targets and drugs obtained from GeneCards (https://www.genecards.org/) and OMIM database (https://www.omim.org/) were imported into Venny 2.1 software to identify the intersecting targets. Venn diagrams were generated to visualize the predicted targets of drug components for disease treatment.

Protein-Protein Interaction Network (PPI) Construction

To identify potential protein-protein interactions (PPI) related to the drug-disease intersection, we employed the String database (https://string-db.org/cgi/input.pl) to construct a PPI network. The constructed network was then imported into Cytoscape 3.6.3 for further analysis using the Network Analyzer tool. Key targets were selected based on 4 parameters: degree, betweenness centrality, average shortest path length, and closeness centrality. Genes with scores higher than the average were considered as key targets and used as nodes to establish an interaction network.

Mouse Models

For homograft murine model, 6-week-old male C57BL/6 mice were from Hangzhou Keyi Experimental Animals Co., Ltd. The animals were housed at the Animal Experimental Research Center of Zhejiang Chinese Medical University. They were kept in a specific pathogen-free (SPF) facility with a temperature of 22 ± 1°C, relative humidity of 40% to 60%, and provided with ad libitum access to food and water. Six-week-old male C57BL/6 mice were randomly assigned to 1 of 5 groups. Each group consisted of 6 mice. A total of 1 × 10⁶ Pan02 cells suspended in 100 μL of PBS were subcutaneously implanted into the right armpit of the mice. The body weights of the mice were recorded, and tumor volumes were calculated every 3 days using the formula 0.5 × length × width ² . After 20 days, all mice were euthanized, and the xenografts were surgically removed and weighed. Histological assessment was performed by staining the samples and conducting IHC analysis. For histological analysis, the samples were stained using IHC staining and evaluated. All animal experiments were conducted in accordance with the ARRIVE guidelines and approved by the Ethics Committee of the Animal Experimental Research Center of Zhejiang Chinese Medical University on September 18, 2023 (IACUC-20220808-08).

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, USA). Data are presented as mean ± standard deviation (SD) from a minimum of 3 independent experiments. Independent group comparisons were conducted using the 1-way analysis of variance (ANOVA). P-value less than .05 was considered statistically significant.

Results

Network Pharmacology Analysis Unveils the Anti-Pancreatic Cancer Function of Tan and Its Potential Mechanism

The main components of Tan were employed to forecast potential targets via the Swiss Target Prediction database. Subsequent to the elimination of duplicates, a total of 214 gene targets were obtained. Cancer-related genes were mined from the GeneCards and NCBI databases, resulting in 1508 targets after deduplication. Comparative analysis using Venny 2.1 revealed 83 overlapping targets between the screened drug and disease targets (Figure 1A), indicating their potential as predictive markers for disease intervention. The interaction network (Figure 1B) between the components of Tan and their respective disease targets was constructed using Cytoscape 3.6.3 software. In this network, the active ingredient of Tan is depicted in blue, while the yellow nodes indicate the targets related to the therapeutic effect of this component against the disease. Further analysis within Cytoscape 3.6.3, utilizing “degree sorting,” pinpointed 10 principal targets based on scores surpassing the average (Figure 1C and D). Node color and size were modulated in accordance to their degree values, with larger and more intensely colored nodes signifying greater degree values. These findings demonstrate the multi-component, multi-gene, and multi-target nature of the anti-tumor effect of Tan.

Figure 1.

Network pharmacology analysis of the anti-pancreatic cancer function and potential mechanisms of Tanshinones. (A) Common targets between Tanshinones targets and pancreatic cancer targets. (B) Target network of 11 active components. The blue hexagon represents the active components, and the yellow rhombus represents the targets. (C) Protein-protein interaction (PPI) network. This network shows the interactions between the active components of Tanshinones and the associated proteins. (D) Core protein network. This network illuminated the pivotal proteins that are intricately involved in the antitumor effects of Tanshinones against pancreatic cancer. The size and color of the nodes depicted their significance within the network, indicating their potential as key regulators in mediating the observed therapeutic outcomes.

Tan Inhibited Cell Viability and Triggered Apoptosis in Pancreatic Cancer Cells

The identification of bioactive constituents present in Tan from SM was accomplished using an HPLC assay. The chromatograms verified the presence of Dihyrotanshinone, Tanshinone I, Cryptotanshinone, and Tanshinone IIA as the predominant constituents (Figure 2A). This finding was consistent with the results obtained from the network pharmacology analysis, confirming these compounds as the major bioactive components in Tan. To evaluate the potential anti-proliferative activity of Tan toward pancreatic cancer cells, we treated PANC-1 and BxPC-3 cells with varying concentrations of Tan for 24 or 48 hours. Utilizing the MTT assay, we observed a dose-dependent and time-dependent reduction in cell viability induced by Tan (Figure 2B). After 48 hours of treatment, the half maximal inhibitory concentration (IC50) values for Tan were determined to be 6.42 μg/mL in PANC-1 cells and 8.09 μg/mL in BxPC-3 cells. In addition to apoptosis, we acknowledge that other mechanisms such as necrosis, autophagy, or potentially senescence could contribute to the reduced cell viability observed. In response to high concentrations of tanshinones in pancreatic cancer cells, we demonstrated an increase in autophagy markers, specifically indicating enhanced autophagic flux as depicted in Supplemental Figure 1A, which shows increased levels of LC3-II and a decrease in p62. Moreover, we have assessed necrosis by measuring lactate dehydrogenase (LDH) release, which is presented in Supplemental Figure 1B. The data indicates an elevation in LDH levels post-treatment with Tan, suggesting that necrotic cell death may also be contributing to the reduced cell viability. To elucidate the apoptotic mechanism of Tan, we conducted DAPI staining and Annexin V-FITC/PI apoptosis Assay. Our findings revealed notable nuclear condensation and fragmentation in both PANC-1 and BxPC-3 cells treated with varying concentrations of Tan, as evidenced by DAPI staining analysis. Notably, treatment with 10 or 30 μg/mL Tan for 48 hours exhibited noticeable nuclear shrinkage and fragmentation, respectively (Figure 2D and F). Moreover, flow cytometric analysis demonstrated a dose-dependent increase of apoptotic cells upon Tan treatment. Compared to the control group, the percentage of apoptotic cells fraction in PANC-1 and BxPC-3 cells was significantly higher (6.2- and 8.8-fold, respectively) after treatment with concentrations close to the IC50 values (Figure 2E and G).

Figure 2.

Tan reduced cell viability and induced cell apoptosis in PANC-1 and BxPC-3 cells. (A) The main components of Tanshinone were identified by HPLC assay. (B, C) PANC-1 and BxPC-3 cells were treated with different concentrations of Tan for 24 and 48 hours, and the cell viability was measured using MTT assay; *P < .05, **P < .01 compared with the control group for 24 hours, ^##P < .01, ^###P < .001 compared with the control group for 48 hours. (D, F) PANC-1 and BxPC-3 cells were treated with Tan for 48 hours, and the nuclei were stained with DAPI and observed by fluorescence microscope. Scale bars are equal to 50 μm. The white arrows indicated fragmented or condensed nuclei. (E, G) Cell apoptosis was assessed by flow cytometry through staining with Annexin V-FITC/PI double.

Tan Activated Caspase Proteins and Decreased Anti-Apoptosis Proteins in Pancreatic Cancer Cells

The levels of apoptosis-related proteins were assessed in both BxPC-3 and PANC-1 cells using Western blot analysis to explore the impact of Tan on apoptosis-related proteins in pancreatic cancer cells. Tan treatment dramatically increased the activity of apoptosis proteins, particularly the cleaved forms of caspase-3 and caspase-9 (Figure 3A and B). Quantitative analysis revealed an increase in cleaved caspase-3 after 30 μg/mL Tan treatment in BxPC-3 cells, with a 2.7-fold amplification compared to the control. Similarly, cleaved caspase-9 levels exhibited a 1.9-fold increase in PANC-1 cells relative to the control, following treatment with Tan at a concentration of 20 μg/mL for 48 hours. Moreover, cleaved PARP levels were also elevated, showing a fold change of 2.8 compared to the control after 20 μg/mL Tan treatment. Additionally, the expression of the antiapoptotic protein survivin was notably reduced in both PANC-1 and BxPC-3 cells upon treatment with Tan. Bcl-2 levels were decreased in BxPC-3 cells, while C-myc levels were decreased in PANC-1 cells. Statistical analysis, using ANOVA, confirmed the significant changes observed. These findings suggest that Tan treatment effectively enhances apoptosis-related protein activity and suppresses the expression of antiapoptotic proteins in pancreatic cancer cells.

Figure 3.

Tan actived caspase proteins and decreased anti-apoptosis proteins in PANC-1 and BxPC-3 cells. (A) BxPC-3 cells were exposed to varying concentrations of Tan for a duration of 48 hours. The protein expression levels of survivin, Cleaved caspase-3, Cleaved caspase-9, and β-actin were detected by Western blotting. Quantitative analysis of the relative expression of the proteins in BxPC-3 cells. The results are represented as mean SEM. Statistical significance was assessed as *P < .1, **P < .01 compared with relative group. (B) PANC-1 cells were treated with different concentrations of Tan for 48 hours. The protein expression levels of Survivin, C-myc, Cleaved caspase-9, Cleaved PARP, β-actin were determined by Western blotting. Quantitative analysis of the relative expression of the proteins in PANC-1 cells. All experiments were repeated a minimum of 3 times. The results are represented as mean SEM. Statistical significance was assessed as *P < 0.1, **P < 0.01 compared with relative group.

Tan Induced Apoptosis by Promoting Excessive ROS in Pancreatic Cancer Cells

ROS are crucial mediators of oxidative stress and an excess of ROS levels contributes to the induction of cancer cell apoptosis. Thus, we hypothesized that Tan induced apoptosis in pancreatic cancer cells by modulating ROS levels. To examine this hypothesis, we investigated ROS levels after treatment with various concentrations of Tan in PANC-1 and BxPC-3 cells. Our results demonstrated a significant increase in ROS levels in both cell lines compared to the control group in a dose-dependent manner. Specifically, the ROS levels of PANC-1 and BxPC-3 significantly increased after treatment with 10 μg/mL Tan for 48 hours (Figure 4A and B). Previous studies have reported that N-acetylcysteine (NAC), an antioxidant, has the ability to restore normal intracellular levels of ROS. To explore the impact of ROS on Tan-induced apoptosis in pancreatic cancer cells, a pre-treatment of 10 mM NAC was administered to PANC-1 cells for 1 hour. To enhance the visualization of ROS activation, we utilized a specialized ROS detection kit and performed observations using an inverted fluorescence microscope. The data demonstrated that Tan, at concentrations of 5 and 10 μg/mL, significantly induced ROS activation in PANC-1 cells. Moreover, the impact of Tan on ROS activation was substantially reduced following pretreatment with NAC (Figure 4C). Quantitative analysis of the fluorescence intensity among various groups was carried out (Figure 4D). Additionally, the induction of apoptosis by Tan via the intrinsic pathway was corroborated by the JC-1 assay. The JC-1 results demonstrated a reduction in the mitochondrial membrane potential of pancreatic cancer cells post-tanshinone treatment, an effect that was reversed in the NAC pretreatment group (Supplemental Figure 2A). Following this, an MTT assay was performed to assess the viability of PANC-1 cells during the subsequent experiment. Additionally, an Annexin V-FITC/PI apoptosis assay was utilized to quantify the percentage of apoptotic cells. Our findings revealed that pre-treatment with NAC improved the cell viability in Tan-treated PANC-1 (Figure 4E) and reversed the apoptotic rates (Figure 4F). Additionally, our examination of apoptosis-related proteins exhibited a reversal in the activation of cleaved PARP and cleaved caspase-9, along with an increased expression level of survivin, compared to cells treated with Tan alone (Figure 4G). Quantitative analysis demonstrated a decrease in the levels of cleaved PARP and cleaved caspase-9 in BxPC-3 cells pre-treated with 10 mM NAC, showing a 13.2- and 1.7-fold amplification, respectively, compared to treatment with 10 μg/mL of Tan alone. Similarly, there was a 1.5-fold increase in survivin levels in PANC-1 cells when pre-treated with NAC, compared with 10 μg/mL of Tan alone (Figure 4H). These findings support the hypothesis that Tan induces apoptosis in pancreatic cancer cells by modulating ROS levels. These findings suggest that Tan induces elevated levels of ROS that cause cellular damage and facilitate cell death. In conclusion, our findings provide compelling evidence supporting the notion that Tan exerts its anti-pancreatic cancer effects by promoting oxidative stress in BxPC-3 and PANC-1 cell.

Figure 4.

Tan induced apoptosis by promoting excessive ROS in pancreatic cancer cells. (A, B) PANC-1 and BxPC-3 cells were treated with 0, 10, or 30 μg/mL Tan for 48 hours. ROS generation was assessed using DCFH-DA probes. The ROS levels for each group were estimated by Flow cytometric assay, and the representative images were presented. (C) ROS production was evaluated using DCFH-DA probes. The ROS concentrations across different groups were determined through the utilization of an inverted fluorescence microscope in PANC-1 cells. (D) A quantitative assessment of ROS relative fluorescence intensity was conducted in PANC-1 cells following treatment with either Tan or NAC for a duration of 24 hours. The results are represented as mean SEM. Statistical significance was assessed as ***P < .001 compared with relative group. #P < .01 compared with the group of 10μg/mL Tan. (E) PANC-1 cells were exposed to different concentrations of Tan, either with or without co-treatment of 10 mM NAC, for a duration of 24 hours. The cell viability was assessed using the MTT assay. (F) The survival rate of PANC-1 cells was assessed using flow cytometry with Annexin V-FITC/PI double staining. Quantitative analysis of the survival rate in PANC-1 and BxPC-3 cells. The results are represented as mean SEM. Statistical significance was assessed as ***P < 0.001 compared with relative group. (G) PANC-1 cells were treated with 10 μg/mL Tan for 48 hours, with or without simultaneous treatment of 10 mM NAC. The protein expression levels of Cleaved PARP, Cleaved caspase-9, survivin, were determined by Western blotting. (H) Quantitative analysis of the expression of protein in PANC-1 cells. The results are represented as mean SEM. Statistical significance was assessed as *P < .05, **P < .01, compared with relative group.

Tan Induced Excessive ROS Not via Inhibition of Nrf2/HO-1 Signaling Pathway

Under oxidative stress conditions, the inactivation ofNrf2/HO-1 signaling is the main regulatory pathway to protect cells against oxidative stress. To investigate the probability of Tan-induced accumulation of ROS via dysregulation of Nrf2/HO-1 in pancreatic cancer cells, we assessed the expression levels of Nrf2 and HO-1 using Western blotting. Our results demonstrated a significant concentration-dependent increase in Nrf2 and HO-1 expression in BxPC-3 and PANC-1 cells treated with varying concentrations of Tan (5, 10, and 20 µg/mL) for 48 hours (Figure 5A and B). We pretreated PANC-1 cells with 10 mM NAC to reduce ROS generation in Tan-treated cells and observed Nrf2 activation and nucleus translocation (Figure 5C). These findings suggest that the increased levels of reactive oxygen species (ROS) observed in pancreatic cancer cells following treatment with Tan are not attributed to the suppression of the Nrf2/HO-1 antioxidant system. Rather, it indicates that the activation of Nrf2/HO-1 signaling is an intracellular response to the increased ROS levels.

Figure 5.

Tan induced excessive ROS ndependently inhibiting the Nrf2/HO-1 signaling pathway. (A, B) PANC-1 and BxPC-3 cells were exposed to varying concentrations of Tan for a duration of 48 hours. Western blot analysis was performed to assess the protein expression levels of Nrf2, HO-1, and β-actin. Quantitative analysis was conducted to determine the relative expression levels of these proteins in PANC-1 and BxPC-3 cells. (C) The representative confocal images of immunofluorescence staining with anti-Nrf2 (red) and DAPI (blue) in PANC-1 cells treated with 10 μg/mL of Tan following a pre-treatment with 10 μM of NAC for 1 hour. Scale bars are equal to 25 μm. All experiments were repeated a minimum of 3 times. The results are represented as mean SEM. The results are shown as mean ± SEM. When comparing the specified groups, statistical significance was determined as *P < .05, **P < .01, ***P < .001, respectively.

Tan Upregulated AKT/FOXO3 Pathway in Pancreatic Cancer Cells

To elucidate the mechanisms by which Tan induces ROS accumulation, we evaluated the activation of AKT/FOXO3, mTOR, and STAT3. Following treatment with different concentrations of Tan for 24 hours, a dose-dependent reduction in the expression of p-STAT3 and mTOR was observed (Figure 6A and B). Interestingly, p-AKT and p-FOXO3 levels were significantly activated after 24 hours of progressive Tan treatment (Figure 6C and D). Quantitative analysis showed that treating cells with 10 µg/mL Tan for 24 hours resulted in a 2.3-fold increase in p-AKT and a 1.4-fold increase in p-FOXO3, while p-STAT3 and p-mTOR decreased by 0.3- and 0.7-fold respectively (P < .05). Further analysis on the time-course activation of the AKT/FOXO3 pathway showed rapid phosphorylation of Akt and FoxO3a in PANC-1 and BxPC-3 cells treated with 10 µg/mL Tan. After 4 hours of treatment, PANC-1 cells demonstrated a significant upregulation inp-Akt, with a fold increase of 3.1. Furthermore, p-FOXO3 exhibited an increase of 1.4-fold, whereas p-STAT3 and p-mTOR decreased by 0.65- and 0.53-fold respectively (Figure 6E and F). Similarly, in BxPC-3 cells, after 6 hours of Tan treatment, p-AKT and p-FOXO3a increased by 2.3- and 1.3-fold respectively, while p-STAT3 and p-mTOR decreased by 0.3- and 0.7-fold respectively (Figure 6G and H). The results obtained from this study provide compelling evidence supporting a pivotal role of the AKT/FOXO3 pathway in the apoptotic effects induced by Tan in pancreatic cancer cells. Subsequent discussion of the implications and significance of these experimental results will undoubtedly contribute to our understanding of the molecular mechanisms underlying Tan-induced apoptosis in this highly malignant disease.

Figure 6.

Tan upregulated AKT/FOXO3 pathway in pancreatic cancer cells. (A-D) PANC-1 cells were treated with various concentrations of Tan for 24 hours. Protein expression levels of mTOR, STAT3, AKT, FoxO3, phosphorylated mTOR, phosphorylated STAT3, phosphorylated AKT, phosphorylated FoxO3, and β-actin were determined using Western blotting. Quantitative analysis was performed to assess the relative expression of these proteins in PANC-1 cells. (E, G) Protein levels of phosphorylated mTOR, phosphorylated STAT3, phosphorylated AKT, phosphorylated FoxO3, and β-actin were determined using Western blotting. PANC-1 and Bxpc-3 cells were treated with 10 μg/mL Tan for different time periods. (F, H) Quantitative analysis was conducted to determine the relative expression of these proteins in PANC-1 and Bxpc-3 cells. All experiments were repeated a minimum of 3 times. The results are expressed as mean ± SEM. Statistical significance was determined as *P < .05, **P < .01, ***P < .001, compared to the indicated groups.

Effect of AKT/FOXO3/SOD2 on ROS Levels

Activation of AKT leads to inactivation of FOXO3, then FOXO3 fails to initiate the transcription of SOD2, leading to ROS accumulation. We hypothesized that Tan treatment would activate the AKT/FOXO3/SOD2 pathway. Indeed, treatment with 5 and 10 µg/mL Tan resulted in increased phosphorylation of AKT and FOXO3 proteins. Furthermore, the expression levels of SOD2 were decreased after various concentrations of Tan. LY294002 is acknowledged as a PI3K/AKT pathway inhibitor, exerting its effects by competitively hindering the lipid kinase functionality of PI3K. This intervention strategically obstructs the initiation of the AKT signaling cascade. ³¹ Therefore, we introduced LY294002 pre-treatment to the cells to investigate the role of the AKT/FOXO3 signaling pathway in enhancing oxidative stress in cancer cells, which subsequently leads to the induction of apoptosis. The results showed that pre-treatment with LY294002 reversed these effects (Figure 7A). Quantitative analysis revealed a significant reduction (P < .05) in p-AKT levels by 2.8-fold and p-FOXO3 levels by 1.5-fold in cells pre-treated with LY294002 after exposure to 10 µg/mL Tan. Moreover, SOD2 levels were increased by 1.5-fold (Figure 7B). These findings suggest that LY294002 inhibits AKT/FOXO3 pathway activation. To further substantiate these findings, experiments were conducted at the mRNA level (Supplemental Figure 3) Interestingly, the addition of LY294002 did not significantly affect the expression of FOXO3 and SOD2 mRNA. This lack of effect on mRNA levels may be attributed to the fact that AKT’s regulatory influence on FOXO3 and SOD2 is predominantly through phosphorylation rather than transcriptional modulation. Furthermore, we aimed to visualize the subcellular localization of FOXO3 protein and its role in ROS regulation. To achieve this, we conducted immunofluorescence (IF) analysis (Figure 7C). Based on our experiments, it was found that treatment with 10 µg/mL Tan induced the translocation of FOXO3 from the nucleus to the cytoplasm, thus suggesting its involvement in the regulation of ROS. The results clearly demonstrated the translocation of FOXO3 from the nucleus to the cytoplasm upon Tan treatment, providing strong evidence to support the role of AKT in ROS regulation through the AKT/FOXO3 pathway. To further investigate the impact of AKT on Tan-induced ROS levels in PANC-1 cells, a pre-treatment with 10 µM LY294002, an AKT inhibitor, was administered. Subsequently, the cells were exposed to 10 µg/mL of Tan for 24 hours. The levels of ROS generation were assessed using a ROS detection kit, and the analysis was conducted using flow cytometry (Figure 7A). The results demonstrated a statistically significant increase in ROS levels following treatment with 10 µg/mL of Tan (P < .05). However, in the presence of the pre-treatment with 10 µM LY294002, this increase in ROS levels was reversed. These findings strongly suggest that inhibiting AKT activity effectively impedes the Tan-induced generation of ROS.

Figure 7.

Effect of AKT/FOXO3/SOD2 on ROS Levels. (A) PANC-1 cells were treated with 5 μg/mL Tan or 10 μg/mL Tan after treated with 10 μM LY294002 for 1 hour. Protein expression levels of phosphorylated STAT3, phosphorylated AKT, phosphorylated FoxO3, SOD2, and β-actin were determined using Western blotting. (B) Quantitative analysis was performed to assess the relative expression of these proteins in PANC-1 cells. (C) PANC-1 cells were treated with 10 μg/mL of Tan following a pre-treatment with 10 μM of LY29002 for 1 hour. Confocal microscopy was utilized to examine the co-localization of DAPI staining and FOXO3 images. Scale bars are equal to 25 μm. (D) PANC-1 were treated with 0 or 10 μg/mL Tan for 48 hours. ROS generation was assessed using DCFH-DA probes. Representative flow cytometric images were obtained, and the ROS levels for each group were presented. (E) Quantitative analysis of the rate of ROS in PANC-1. All experiments were repeated a minimum of 3 times. The results are shown as mean SEM. When comparing control group, statistical significance was determined as **P < .01, respectively.

The Positive Feedback Effect of ROS on AKT/FOXO3/SOD2 in PANC-1 Cells

To explore the positive feedback effect of ROS on the AKT/FOXO3/SOD2 signaling pathway in pancreatic cancer cells, PANC-1 cells were pre-treated with the ROS scavenger NAC for 1 hour before incubation with different concentrations of Tan. The effects of NAC pre-treatment on the AKT/FOXO3/SOD2 pathway were assessed using Western blot analysis. As depicted in Figure 8A, pre-treatment with NAC effectively reversed the Tan-induced activation of the AKT/FOXO3/SOD2 pathway. Quantitative analysis revealed a significant 4.3-fold increase in p-AKT and a 1.4-fold increase in p-FOXO3 after 24 hours of treatment with 10 µg/mL Tan (P < .05). However, NAC pre-treatment alleviated this effect. Furthermore, the expression level of SOD2, a crucial enzymatic ROS scavenger, was elevated by 1.8-fold compared to the group treated solely with 10 µg/mL Tan for 48 hours (P < .05), as shown in Figure 8B. Additionally, immunofluorescence analysis demonstrated that NAC treatment significantly enhanced the levels of FOXO3 protein and facilitated its translocation into the nucleus, compared to cells treated with Tan alone (Figure 8C). Overall, these findings suggest that Tan induces the production of intracellular ROS, thereby activating the AKT/FOXO3 signaling pathway and downregulating SOD2 expression. Consequently, the accumulation of ROS ultimately sensitizes pancreatic cancer cells to ROS-mediated apoptosis. This highlights the potential therapeutic strategy of selectively eliminating cancer cells through Akt activation. Future studies should focus on investigating the downstream targets of the AKT/FOXO3/SOD2 pathway and elucidating the mechanisms by which ROS regulate their expression. Such investigations will enhance our comprehensive understanding of the therapeutic potential and limitations of targeting ROS and the AKT/FOXO3/SOD2 pathway in the treatment of pancreatic cancer.

Figure 8.

The positive feedback effect of ROS on AKT/FOXO3/SOD2 in PANC-1 cells. (A) PANC-1 cells were treated with 5 μg/mL Tan or 10 μg/mL Tan after treated with 10 μM NAC for 1 hour. The protein expression levels of Cleaved caspase-9, Survivn, Cleaved PARP, FoxO3, and β-actin were determined by Western blotting. (B) Quantitative analysis of the relative expression of the proteins in PANC-1 cells. (C) PANC-1 cells were treated with 10 μg/mL of Tan following a pre-treatment with 10 μM of NAC for 1 hour. Confocal microscopy was utilized to examine the co-localization of DAPI staining and FOXO3 images. Scale bars are equal to 25 μm. All experiments were repeated a minimum of 3 times. The results are shown as mean SEM. When comparing the specified groups, statistical significance was determined as *P < .05, **P < .01, respectively.

The Antitumor Effects of Tan In vivo

To explore the in vivo anticancer properties of Tan further, we conducted a study utilizing a subcutaneous tumor model in C57BL/6 mice. The mice were administered daily oral doses of Tan at 10, 20, and 30 mg/kg, or in conjunction with intraperitoneal injections of gemcitabine (30 mg/kg) every 3 days. Tumor size was measured and body weight was recorded every 3 days. On day 19, the subcutaneous tumors were surgically removed and photographed. Our findings demonstrated that Tan intervention at all doses significantly reduced tumor size, along with the notable effect observed with gemcitabine treatment. Specifically, the blank control group increased from an initial volume of 108.21 to 4747.63 mm³, while the high-dose Tan group increased from an initial volume of 123.46 to 3435.50 mm³, resulting in a tumor inhibition rate of 25.697% (Figure 9A and B). To gain insights into the potential mechanisms underlying antitumor activity of Tan, we conducted Western blot analysis to examine critical signaling pathways. Our results revealed that both Tan and gemcitabine treatment activated the AKT/FOXO3/SOD2 signaling pathway, consistent with our findings from cell studies. As depicted in Figure 9C, the expression levels of p-AKT exhibited a significant increase of 1.5-fold in the high-dose Tan group, while the gemcitabine group experienced a 1.5-fold increase. Additionally, the high-dose Tan group exhibited a decrease of 0.7-fold in FOXO3 expression and a 0.5-fold decrease in SOD2 compared to the control group. Immunohistochemical analysis further demonstrated a significant decrease in PCNA-positive and survivin-positive cells following Tan and gemcitabine intervention. Overall, our study indicates that Tan possesses remarkable in vivo antitumor properties, exerting its effects through the activation of the AKT/FOXO3/SOD2 signaling pathway. These findings shed light on the potential of Tan as a natural compound for the treatment of pancreatic cancer, opening up promising avenues for further research and development.

Figure 9.

The antitumor effects of Tan in vivo. (A, B) Pan02 cells 1 × 106 were injected subcutaneously into C57BL/6 mice. Tumor growth and burden were assessed at specified time intervals. (C) The protein levels of p-AKT, FoxO3, SOD2, and GAPDH were determined by Western blotting. (D) Quantitative analysis of the relative expression of the proteins in tumor. (E) The images displayed depict representative serial sections of xenograft tumor samples subjected to immunohistochemical staining for PCNA and survivin. The results are expressed as mean ± SEM. Statistical significance was determined as ***P < .001, compared to the indicated groups.

Discussion

Pancreatic cancer (PC) is an exceptionally aggressive and malignant solid tumor with limited treatment options and a dismal prognosis. ² Although chemotherapeutics offer modest survival benefits, they can also lead to severe adverse reactions, including liver and renal toxicity, gastrointestinal bleeding, and cardiovascular toxicity. ³² In recent years, there has been a growing interest in natural medicine, particularly traditional Chinese medicine (TCM). TCM has gained attention due to its well-established medicinal properties, such as good therapeutic effects, low toxicity, and minimal side effects. This has attracted researchers to explore its potential application in the field of cancer treatment. Increasing evidence from various studies has shown that TCM contains multiple compounds with anticancer activity, opening new avenues for cancer research. For example, hernandezine, a bisbenzylisoquinoline alkaloid extracted from Thalictrum glandulosissimum, has been found to induce autophagy in pancreatic cancer cells through the activation of the ROS/MAPK signaling pathway. ¹⁰ In addition, another study demonstrated that the ethanol extract from Cyperus exaltatus var. iwasakii exerts anti-prostate cancer effects both in vitro and in vivo by inhibiting the cell cycle and reducing MMP-9-induced metastasis. ³³ Danshen (Salvia miltiorrhiza Bunge), a traditional Chinese medicine, has been utilized for centuries in the treatment of coronary artery disease and cerebrovascular diseases. ³⁴ The liposoluble ingredients extracted from the roots and rhizomes of Danshen, known as Tanshinones (Tan), have exhibited various biological functions, including anti-inflammatory, anticoagulant, and antibacterial effects. The anticancer activity of Tan been confirmed in colorectal cancer and liver cancer.^24-26 In this study, we investigated the potential anti-pancreatic cancer effects of Tan and its active components. By employing network pharmacology analysis, we identified multiple active components within Tan that showed promise as potential anticancer agents against pancreatic cancer. Our analysis incorporated consideration of multi-components, multi-genes, and multi-targets, reflecting the intricate effect of Tan on anticancer effect (Figure 1A-D). To further explore the bioactive ingredients, we performed HPLC assays. The chromatograms revealed Dihyrotanshinone, Tanshinone I, Cryptotanshinone, and Tanshinone IIA as the major constituents of the Danshen-derived Tan extract (Figure 3A). In conclusion, our study highlights the potential of Tan and its active components as alternative therapeutic options for treating pancreatic cancer.

The clinical management of cancer has long prioritized strategies targeting programmed cell death or apoptosis as an effective means to eliminate cancer cells. ⁵ Caspases, a family of cysteine proteases, play crucial roles in regulating apoptosis. ³⁵ Previous studies have demonstrated a strong correlation between the fraction of apoptotic cells and overall survival in pancreatic ductal adenocarcinoma (PDAC), highlighting the significant role of the apoptotic machinery in the tumor biology of this disease.^36,37 Despite the importance of apoptosis in PDAC, limited research has been conducted on the anti-cancer activity and mechanism of action of Tan in pancreatic cancer. In our study, we utilized MTT and flow cytometry assays to examine the impact of tanshinones on the growth of pancreatic cancer cells in a controlled laboratory environment. Our findings revealed a remarkable suppression of cell proliferation and the initiation of cell death in pancreatic cancer cells (Figure 2B-G). Moreover, through the application of DAPI staining, we observed distinct evidence of nuclear fragmentation and increased condensation of chromatin following the administration of Tan. In order to gain a deeper understanding of the molecular mechanisms underlying Tan-induced apoptosis, our investigation centered on the analysis of apoptosis-related protein expression. Western blot analysis demonstrated that Tan treatment resulted in an upregulation of the expression of cleaved caspase-3 and cleaved caspase-9, which are critical mediators of the apoptotic pathway (Figure 3A and B). Moreover, treatment with Tan resulted in the downregulation of anti-apoptotic proteins including survivin, Bcl-2, and c-myc in the treated cells. These findings suggest that Tan exerts its anti-cancer effects in pancreatic cancer cells through the modulation of apoptosis-related protein expression. In conclusion, our study provides preliminary evidence for the anti-cancer activity of Tan in pancreatic cancer cells through the induction of apoptosis and modulation of apoptosis-related protein expression.

Reactive oxygen species (ROS) are metabolites that induce oxidative stress in cells and can trigger apoptosis when produced excessively. ³⁸ Previous pharmacological studies have highlighted the importance of ROS in programmed cell death and apoptosis induction. ³⁹ Interestingly, tumor cells often exhibit abnormally high levels of ROS, rendering them more susceptible to oxidative stress. ⁴⁰ This suggests that interfering with antioxidant pathways or increasing ROS burden may selectively kill cancer cells. Our findings demonstrated a significant dose-dependent increase in ROS generation in response to Tan treatment in both cell lines (Figure 4A-D). To validate the effects of ROS on apoptosis in pancreatic cancer cells, we employed N-acetyl cysteine (NAC), a potent ROS scavenger, to pre-treat PANC-1 cells prior to Tan treatment at concentrations of 5 and 10 µg/mL. Consistent with our hypothesis, NAC pre-treatment led to a decrease in apoptosis protein expression compared to cells without NAC pre-treatment (Figure 4E and F). Apoptosis-related protein levels were assessed using western blotting (Figure 5G and H).

The current investigation sought to explore the role of the Nrf2/HO-1 pathway in the cellular response to Tan treatment in pancreatic cancer cells. Our findings have unveiled an intriguing revelation, wherein the activation of the Nrf2/HO-1 pathway seemed to occur autonomously, regardless of the Tan treatment (Figure 5A and B). The complex interplay between reactive oxygen species (ROS) production, Nrf2-mediated antioxidant responses, and cell death pathways is a subject of great interest in cancer research. Previous studies have demonstrated that initial ROS production induced by anti-cancer drugs can activate the Nrf2/HO-1 pathway in cancer cells, providing a survival mechanism by mitigating oxidative damage. However, it is noteworthy that when ROS levels exceed the cellular antioxidant capacity, a tipping point is reached, leading to irreversible oxidative damage and subsequent cell death. Although the activation of the Nrf2 pathway is a survival mechanism, excessive ROS levels can overcome this pathway and induce cytotoxicity. Interestingly, our investigation showed that Tan treatment induces ROS generation. To confirm the role of ROS in Nrf2 activation, we employed the ROS scavenger N-acetyl cysteine (NAC). Immunofluorescence analysis revealed that NAC effectively reversed the activation and nuclear translocation of Nrf2 in Tan-treated cells, implicating the role of ROS in Nrf2 activation (Figure 5C). In conclusion, understanding the complex interplay between ROS generation, Nrf2-mediated antioxidant responses, and cell death pathways in the context of Tan treatment in pancreatic cancer cells can pave the way for the development of more effective therapeutic strategies for this disease.

In human cancers, AKT is commonly activated and often considered an oncoprotein. This activation can occur through various mechanisms, such as PTEN mutation and Ras activation. ¹¹ However, it should be noted that pan-Akt inhibition can have adverse consequences, such as hyperglycemia, hyperinsulinemia, and diabetes.^12,13 Interestingly, despite its ability to inhibit apoptosis, hyperactivated AKT did not protect against ROS-induced cell death but rather sensitized cells to this mechanism of cell death. This finding suggests that hyperactivated AKT may serve as the Achilles’ heel of Akt, leading to potential therapeutic opportunities for selectively targeting and killing cancer cells. ¹² Thus, a desirable therapeutic approach would be to selectively target cancer cells displaying hyperactive AKT signaling, which could minimize off-target effects and enhance treatment efficiency. Previous studies have reported that hyperactivated AKT attenuates G2 arrest in Rat1a cells following DNA damage and promotes apoptosis of hepatocellular carcinoma cells through the activation of the Akt/FoxO3 signaling pathway and increased intracellular ROS levels induced by Juglanthraquinone C treatment. ¹⁸ In this study, we aimed to investigate the effects of hyperactivated AKT on pancreatic cancer cells apoptosis. Our results demonstrated that hyperactivated Akt induces sensitizes pancreatic cancer cells to ROS-mediated apoptosis. This effect is possibly mediated by increased intracellular ROS levels due to enhanced oxygen consumption and the inhibition of ROS scavenger expression downstream of the transcription factor FOXO. Our study demonstrated that treatment with Tan activated the AKT/FOXO3/SOD2 pathway in a time and dose-dependent manner (Figure 6). Importantly, the activation of this signaling pathway and the generation of ROS could be reversed by pretreating cells with the AKT inhibitor LY294002 (Figure 7). To further investigate the role of ROS in AKT/FOXO3/SOD2 pathway, we treated cells with the ROS scavenger N-acetylcysteine (NAC). Our results showed that the activation of AKT/FOXO3/SOD2 could be reversed by NAC treatment (Figure 8). Furthermore, we have confirmed the anti-growth effects of Tan on pancreatic cancer in vivo (Figure 9). Through Western blot analysis, we have validated that Tan and gemcitabine exert their anti-pancreatic cancer effects by activating the AKT/FOXO3/SOD2 pathway. The immunohistochemical results have also revealed a noteworthy reduction in the number of PCNA and survivin-positive cells after treatment with Tan and gemcitabine. In summary, our study highlights the potential therapeutic value of targeting hyperactivated AKT signaling in cancer cells. Our findings demonstrate that hyperactivated AKT renders pancreatic cancer cells more susceptible to apoptosis induced by ROS. Additionally, we observed that the activation of the AKT/FOXO3/SOD2 pathway is contingent upon the levels of ROS, highlighting the pivotal role of ROS regulation in both cancer cell survival and therapy.

In conclusion, our study employed pharmacology network analysis to identify the key constituents of Tan and their specific targets in the therapeutic approach for pancreatic cancer. We found that Tan exerts its apoptotic effects on pancreatic cancer cells through the activation of an ROS-dependent pathway. This process is mediated by a positive feedback loop mechanism, wherein Tan activates the AKT/FOXO3/SOD2 pathway, resulting in intracellular ROS accumulation. The accumulated ROS further activates the AKT/FOXO3/SOD2 pathway, leading to the generation of more ROS and subsequent apoptosis of pancreatic cancer cells. Furthermore, we successfully validated the efficacy of Tan in inhibiting pancreatic tumor growth using a subcutaneous syngeneic transplant tumor model in C57BL/6 mice. Our findings, supported by Western blotting and immunohistochemistry, revealed that Tan inhibits pancreatic cancer tumor growth by activating the AKT/FOXO3/SOD2 pathway. These findings have significant implications for the treatment of pancreatic cancer. The selectivity of Tan toward pancreatic cancer cells with hyperactivated AKT pathway can help spare normal cells, minimizing potential side effects and improving patient tolerance to the treatment. Moreover, the positive feedback loop mechanism uncovered in this study adds novelty to the understanding of Tan's anti-cancer properties. The identification of the AKT/FOXO3/SOD2 pathway as a crucial mediator in Tan-induced apoptosis provides an avenue for further research and potentially novel therapeutic interventions in pancreatic cancer. Overall, our study demonstrates the effectiveness and selectivity of Tan in targeting and killing pancreatic cancer cells. These findings provide important insights into the molecular mechanisms driving pancreatic cancer progression and offer potential avenues for the development of targeted therapies using natural compounds.

Supplemental Material

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Supplemental material, sj-docx-4-ict-10.1177_15347354241258961 for The Tanshinones (Tan) Extract From Salvia miltiorrhiza Bunge Induces ROS-Dependent Apoptosis in Pancreatic Cancer via AKT Hyperactivation-Mediated FOXO3/SOD2 Signaling by Qin Xu, Shujie Dong, Qiuyi Gong, Qun Dai, Rubin Cheng and Yuqing Ge in Integrative Cancer Therapies