Andrographolide enhances the antitumor activity of cisplatin by inhibiting the PI3K/AKT signaling pathway in bladder cancer

doi:10.21203/rs.3.rs-4651936/v1

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Andrographolide enhances the antitumor activity of cisplatin by inhibiting the PI3K/AKT signaling pathway in bladder cancer

https://doi.org/10.21203/rs.3.rs-4651936/v1

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Bladder cancer (BC) is the most common malignant tumor of urinary system worldwide. Resistance to cisplatin(CDDP) in advanced bladder cancer leads to reduced survival rates. Andrographolide(Andro), an active ingredient extracted from Andrographis paniculate, has been reported to have multidimensional pharmacological properties. Here we report the therapeutic effect of Andro and CDDP on BC. BIU87 Cisplatin resistant (BIU87-CISR) cells were exposed to Andro or CDDP in different concentrations in vitro. In vivo, the subcutaneous tumor bearing (BIU87-CisR cells) mouse model was constructed. The results indicated combination therapy of Andro and CDDP significantly inhibited BIU87-CisR cells proliferation, migration and invasion. Andro treatment alleviated epithelial-to-mesenchymal transition (EMT) in BIU87-CisR cells. Furthermore, the combination treatment enhanced inhibition of Andro on PI3K/AKT signaling pathway. The molecular docking was also used to support the above results. Taken together, Andro enhanced the effect of CDDP therapy by modulating PI3K/AKT signaling pathway to reduce EMT in cisplatin resistant bladder cancer cells. This study provides a theoretical basis for the clinical application of Andro as an adjuvant drug in the treatment of BC.

Biological sciences/Cancer

Health sciences/Diseases

Health sciences/Urology

andrographolide

bladder cancer

cisplatin

chemotherapy resistance

PI3K/AKT

Bladder cancer (BC) is the most prevalent malignant tumor of the urinary system worldwide, with the highest incidence and mortality rate of all urinary tumors in China [1]. Cisplatin-based chemotherapy is a standardized treatment regimen for patients with muscle-invasive bladder cancer (MIBC) [2]. However, the development of cisplatin (CDDP) resistance ultimately results in treatment failure and contributes to an escalating death rate. Approximately 40% of BC patients have acquired drug resistance during their disease progression [3]. The molecular mechanism of cisplatin resistance has not been fully elucidated. Therefore, it is urgent to investigate therapeutic approaches to overcome cisplatin resistance in BC patients.

The role of epithelial-mesenchymal transition (EMT) in acquired resistance to chemotherapy is an emerging research field. Current studies have shown that EMT is involved in the initial process of tumor metastasis [4]. The occurrence of EMT implies that the tumor has acquired higher invasion and immune escape capacities [5]. Studies have shown that EMT-related markers are upregulated in MIBC [6]. Furthermore, the expression of mesenchymal cell markers (N-cadherin and Vimentin) was positively correlated with the disease severity and poor prognosis [7]. Hence, targeting EMT in BC could be a promising treatment option.

Plant-derived compounds, such as taxol and vincristine, have been used clinically as first-line antitumor drugs [8]. Andrographis paniculate, a traditional medicinal plant, is widely used to treat snake and insect bites, diabetes, dysentery, malaria, and other diseases [9, 10]. Andrographolide (Andro)—the main bioactive chemical component extracted from Andrographis paniculate—has been shown to exert significant antitumor activity [11]. Apoptosis induction, cell cycle arrest, anti-angiogenesis, and regulation of the immune macroenvironment comprise the underlying mechanism by which Andro suppresses tumor growth [12]. According to various studies, Andro has been found to inhibit the growth of colon cancer cells by enhancing cisplatin-induced endoplasmic reticulum stress and inhibiting STAT3 expression [13]. It has also been observed to enhance TRAIL-induced apoptosis in human gastric cancer cells by inducing DR5 expression, which is related to the production of ROS in tumor cells[14]. Furthermore, Andro has been shown to induce cycle arrest and apoptosis in pancreatic cancer cells by inhibiting the activation of STAT3 and Akt. When combined with gemcitabine treatment, Andro has a more significant inhibitory effect on cell growth compared to single treatment [15]. Moreover, Andro induces autophagy in osteosarcoma cells by inhibiting the PI3K/Akt/mTOR pathway and enhancing the JNK signaling pathway [16]. In a study by Li et al., it was confirmed that Andro inhibits the expression of miR-21-5p by suppressing NF-κB, thereby potentially exerting an anti-cancer effect on metastatic breast cancer [17]. Additionally, in non-small cell lung cancer, Andro reduces the expression of PD-L1 by inhibiting JAK2/STAT3 signaling [18]. These findings collectively suggest that Andro exhibits significant anti-tumor activity.

Our previous study found that Andro inhibits the growth of BC by inhibiting NF-κB and PI3K [19]. However, the role of Andro in CDDP-resistant BC needs further inquiry. In our study, the cisplatin—Andro combination significantly inhibited the proliferation, migration, and invasion of BIU87-CisR cells. Andro treatment upregulates E-cadherin expression and downregulates N-cadherin and vimentin expression by inhibiting the PI3K/Akt signaling pathway. This study provides a theoretical basis for the clinical application of Andro as an adjuvant drug in treating BC.

Chemicals and antibodies

Andrographolide (Andro, > 97% purity) was obtained from Sigma-Aldrich Chemical Co. and dissolved in DMSO. The primary antibodies—anti-E-cadherin, N-cadherin, Vimentin, p-AKT, AKT, p-PI3K, PI3K, and GAPDH—were obtained from Abclonal (Wuhan, China). The secondary antibodies—goat anti-mouse IgG HRP or goat anti-rabbit IgG HRP—were obtained from Proteintech (Wuhan, China).

Cell culture

The BIU87 and BiU87-CISR human BC cell lines were obtained from Shanghai Fuheng Biotechnology Co., LTD. and cultured in incubators containing 5% CO₂ at 37℃ with RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin.

Detection of cell proliferation capacity

Cell proliferation ability was assessed using the CCK8 colony formation assay. For the CCK8 assay, 5000 cells/well were inoculated into 96-well plates. Parallel groups were set up for the experimental and control groups. Cell viability was measured 24, 48, and 72 h after stimulation using the CCK-8 kit. Following the preceding steps, 10 µL of CCK-8 solution was added to each hole of the 96-well plate, and the plate was incubated for 2 h. The absorbance value at 450 nm was measured using a microplate reader (Bio-Rad). For the colony formation assay, 250 cells/well were seeded in 6-well plates and incubated for 24 h. The cells were treated according to experimental groups for an additional 2 weeks. The medium was discarded, washed twice with PBS, and then fixed with 4% paraformaldehyde for 20 min. Crystal violet solution was used for staining the cells. Photographs were then taken, and clones were counted.

Wound healing assay

The cells in the logarithmic growth phase were digested and counted. The cells were diluted by multiples and seeded in a 6-well plate at a density of 5 × 10⁵ cells/well. The plate was gently shaken to disperse the cells evenly. Once the cells adhered to the wall and formed a monolayer overnight, a 200 µL pipette tip was used to carefully scratch the monolayer cells, with the assistance of a sterilized ruler to ensure a vertical and non-tilted position of the pipette tip. The scratched cells were then washed multiple times with PBS to remove them, followed by the addition of serum-free medium. Different concentrations of andrographolide were applied and the cells were cultured for 24 hours after adding the drug. The healing distance of the scratch was observed and photos were taken.

Cell apoptotic ratio analysis

The FACS Calibur flow cytometer (Beckman Coulter) was used to analyze apoptotic cells. Cells from different groups were collected and washed twice with cold PBS. The cells were suspended with 400 µL 1× binding buffer and added with AnnexinV-FITC (5 µL). After mixing, the cells were incubated at 2℃-8℃ and protected from light for 15 min. Approximately 10 µL of PI was added and incubated for another 5 min under the same condition. The staining was detected by flow cytometry within 1 h.

Cell immunofluorescence

In brief, different groups of cell climbing slices were washed with PBS, fixed with 4% paraformaldehyde, and blocked with 10% goat serum for 30 min. Sufficient amounts of primary antibodies were incubated overnight on coverslips, washed three times in PBS, and then incubated for 2 h at 25°C with secondary antibodies. The slides were observed with a fluorescence microscope (Olympus, Tokyo, Japan).

Western Blot

1× RIPA buffer (Wanleibio, Beijing, China) was used for extracting protein from cells and tumor tissues. The BCA protein quantification kit (Beyotime, Shanghai, China) was used for protein quantification. Approximately 30 µg of protein samples were loaded and electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to NC membranes. The membranes were blocked for 1 h in 5% milk powder and then incubated with primary antibodies overnight at 4°C. The next day, the membranes were incubated with the appropriate secondary antibody for 1 h after washing in PBST. A Protein Simple chemiluminescent imager was used for imaging the membranes through enhanced chemiluminescence (ECL) western blotting substrate.

Molecular docking

The 3D structure file of the target protein was downloaded from the PDB database. Pymol software was used for deleting water molecules and small molecular ligands from the obtained structure. Following the processing steps, the protein receptor molecular structure file is obtained. The 2D structure file of Andro was downloaded from the PubChem database. Chem3d was used to generate a 3D structure from a 2D structure file. The Autodock software was used to hydrogenate the protein receptor file, and the small-molecule ligand file was also converted into a format recognizable for molecular docking. The active pockets of protein receptors were determined to obtain the location and size parameters of the "grid box". Finally, Vina software was used to perform molecular docking.

Animal experiments

A total of 12 Balb/c male nude mice (6-week-old) with an average weight of 20 g were obtained from Liaoning Changsheng Co., LTD. The mice were kept in SPF conditions at 24°C ± 2°C, 45% humidity, and provided plenty of sterilized food and water. The animal experiments were approved by the Animal Ethics Committee of the First Hospital of Jilin University.

The mice were randomly divided into four experimental groups: Andro, CDDP, combination, and control. BIU87-CisR cells in the logarithmic growth phase were collected, counted, and cleaned with PBS. The cells were injected into the subcutaneous part of the armpit of mice (5 × 10⁶ cells/flank). The tumor volume was evaluated with vernier calipers every 3 days after injection, Tumor volume (mm³) = maximum length × vertical width²/2. On the 6th day of tumor-bearing, mice were given treatment (CDDP at a dose of 2 mg/kg, and Andro at a dose of 10 mg/kg), The specific treatment plan involves administering an intraperitoneal injection of Andro once every 3 days for a total of 6 times in the Andro monotherapy group. In the CDDP monotherapy group, an intraperitoneal injection of CDDP will be given once every 3 days. The combined treatment group will receive an intraperitoneal injection of both Andro and CDDP once every 3 days. Tumors were isolated after mice were euthanized. For euthanasia, mice were injected with overdose of pentobarbital intraperitoneally.

Statistical analysis

All data were analyzed using GraphPad Prism 9 (La Jolla, CA, USA). The differences between the data sets were analyzed using the t-test or one-way analysis of variance (ANOVA). P value < 0.05 was considered statistically significant.

Biu87-CisR cells exhibited a mesenchymal phenotype and CDDP resistance

To explore the differences in EMT phenotypes between common BC cells and cisplatin-resistant BC cells, we used immunofluorescence staining and western blot (WB) to detect the expression levels of EMT-related proteins in BIU87 and BiU87-CisR cell lines. Fluorescence staining showed that the intensity of green fluorescence expressed by E-cadherin on the cell membrane of BiU87-CisR was weaker compared with that of BIU87 (Fig. 1A). WB analysis suggested that Vimentin and N-cadherin expression were increased in Biu87-CisR cells, while E-cadherin protein expression was decreased (Fig. 1B). These findings suggest that BIU87-CisR cells have undergone an EMT process.

The cell viability under cisplatin or Andro stimulation for 24h was analyzed using CCK-8. Under the same concentration of CDDP, BIU87-CisR cells showed obvious resistance. However, the viability of Biu87 and Biu87-CisR cells showed no significant difference at several concentrations of Andro (Fig. 1C). Furthermore, 50% inhibition (IC50) of CDDP against BIU87 and Biu87-CisR cells was 2.808 µM and 5.848 µM, respectively, and the IC50 of Andro against BIU87 and Biu87-CisR cells was 29.29 µM and 32.93 µM, respectively.

Andro suppressed EMT in Biu87-CisR cells

To investigate the effect of Andro on EMT phenotype in BIU87-CisR cells, we stimulated the cells with Andro at varying concentrations of 1, 3, and 6 µM for 72 h. Then, As shown in Fig. 2A, compared with the control group, the fluorescence intensity of E-cadherin gradually enhanced as the Andro concentration increased. WB suggested that Andro treatment increased the expression of E-cadherin in BIU87-CisR cells and decreased the expression of Vimentin and N-cadherin. The comparison of gray values was statistically significant (Fig. 2B). The results demonstrated that Andro inhibited EMT in cisplatin-resistant BC cells.

Andro enhances CDDP-induced inhibition of proliferation, colony formation, and migration in Biu87-CisR cells

In this study, BIU87-CisR cells were treated with CDDP (0.3 µM) and/or Andro (3 µM) for 72 h, and cell viability was detected using CCK-8. As shown in Fig. 3A, the proliferation of Biu87-CisR cells in the CDDP and Andro group was significantly inhibited compared with that in CDDP or Andro alone. The clone formation assays also demonstrated that the combination treatment significantly restrained the clone formation capability of Biu87-CisR cells (Fig. 3B).

The scratch assay revealed that the combination treatment could inhibit scratch healing compared with treatment with Andro or CDDP alone (Fig. 3C). Transwell migration and invasion assay showed that the BIU87-CisR cells migration ratio was decreased by CDDP but not significantly. Andro—CDDP combination could significantly inhibit the migration and invasion ability of BIU87-CisR cells (Fig. 3D), the difference was statistically significant (P < 0.01). The MMP-9 expression from different conditions was tested; combined administration effectively inhibited MMP-9 (Fig. 3E). In conclusion, BIU87-CisR cells showed poor responsiveness to Cisplatin treatment, while the proliferation, migration, and invasion of Biu87-CisR cells decreased significantly following the co-administration of Andro. Therefore, Andro might have a synergistic effect with Cisplatin in BIU87-CisR cells.

Andro synergistically enhances CDDP-induced apoptosis in Biu87-CisR cells

To detect the effect of CDDP and/or Andro on the apoptosis of BIU87-CisR cells, we treated BIU87-CisR cells with a final concentration of 3 µM CDDP and/or 30 µM Andro. Subsequently, flow cytometry was used to detect cell apoptosis. The results are shown in Fig. 4A. CDDP treatment alone could slightly induce apoptosis. The apoptosis rate of the combination drug group was significantly higher than that of the single drug group; the difference was statistically significant. Our previous study revealed that Andro induced endogenous apoptosis in BC cells via the mitochondrial pathway [19]. Meanwhile, we also detected the expression of cleaved caspase-3, Bax, and Bcl2. No significant changes in protein expression were observed in any monotherapy group; combination therapy significantly increased Bax, cleaved caspase-3 expression, and decreased Bcl2 expression (Fig. 4B).

Treatment with the Andro—CDDP combination reversed the EMT process in Biu87-CisR cells

The aforementioned studies indicated that BIU87-CisR cells underwent the EMT process, and Andro alone inhibited EMT in BIU87-CisR cells in a dose-dependent manner. EMT plays an important role in the occurrence of Cisplatin resistance in BC. To further explore the mechanism of the antitumor effect of Andro and Cisplatin, we used WB to detect the expression of EMT-related proteins in BIU87-CisR cells treated with Andro and/or CDDP. The results showed that, compared with the Cisplatin or Andro group, combination treatment had a more pronounced inhibitory effect on EMT, evidenced by a significant decrease in the expression of Vimentin and N-cadherin and an increase in the expression of E-cadherin (Fig. 5).

Andro—CDDP combination inhibited the PI3K/AKT pathway in Biu87-CisR cells

The EMT mechanism is reversible and can be regulated through the PI3K signaling pathway [20]. Phosphorylated AKT inhibits Glycogen synthase kinase 3 (GSK-3), whose activity is critical for increasing E-cadherin levels [21]. As shown in Fig. 6A, Andro treatment alone down-regulated the expression of p-AKT and p-PI3K in BIU87-CisR cells without affecting their non-phosphorylated forms. However, the inhibitory effect of the combination drug was significantly better than that of either drug alone. Next, we used AutoDock Vina for molecular docking to verify whether Andro can directly regulate the PI3K/AKT pathway. The results demonstrated that Andro could bind to five amino acids—LYS276, LEU295, SER7, LYS179, and PHE161—in the AKT active center with the lowest free energy among the 20 simulated docking modes. Furthermore, Andro could bind to TRP201 in the PI3K active center (Figs. 6B&C).

Effect of Andro combined with CDDP on mice bearing BIU87-CisR cell

To investigate the effect of Andro combined with CDDP on the growth of BC in vivo, we inoculated BIU87-CisR cells into the armpit of mice. The results showed that the combination therapy significantly suppressed the growth of tumors (Figs. 7A-C). We then examined the expression of EMT and PI3K pathways. The results were consistent with those of in vitro experiments. Compared with the Cisplatin or Andro groups, the combination treatment inhibited the expression of Vimentin and N-cadherin. For the PI3K pathway, the p-AKT, p-PI3K, and p-mTOR protein levels in tumor tissues from the combination group were lower than in the monotherapy groups (Figs. 7D-E). These findings suggest that Andro could be a PI3K/AKT inhibitor to alleviate the EMT process in BIU87-CisR cells, These findings suggest that Andro may act as a PI3K/AKT inhibitor to alleviate the EMT process in BIU87-CisR cells. Furthermore, when compared to the CDDP monotherapy group, the combination of Andro and CDDP significantly inhibited tumor growth.

Chemotherapy is the first-line treatment for advanced tumors. However, chemotherapy resistance is an increasing challenge to doctors. CDDP-based combinations are usually used as standard regimens for advanced BC. Therefore, overcoming cisplatin resistance is an urgent issue. The present study demonstrated that Andro alleviated EMT in CDDP-resistant BC cells by regulating the PI3K/AKT signaling pathway and enhances the antitumor activity of CDDP.

The occurrence of EMT implies that tumor cells acquire higher invasion characteristics, immunosuppression, stem-like characteristics, and resistance to chemotherapy-induced apoptosis [22]. The effects of CDDP chemotherapy are associated with increased migration ability of classically activated macrophage (CAM), and studies have shown that induction of CAM by Cisplatin triggers the EMT mechanism [23, 24]. Chemokine ligand 20 (CCL20) can recruit T helper cells to maintain an immunosuppressant microenvironment and ensure the survival of cancer cells [25, 26], whereas CDDP chemotherapy stimulates the secretion of CCL20 from CAM. The CCL20/CCR6 axis enhances tumor cell migration and induces EMT mechanisms, resulting in drug resistance [27].

We established BIU87-CisR cells by gradually increasing CDDP concentration, confirming that long-term use of CDDP would lead to the occurrence of drug resistance. First, we found that Biu87-CisR cells were not only less sensitive to Cisplatin stimulation but also had higher expression levels of N-cadherin and Vimentin than BIU87 cells. Recent studies have shown that Andro inhibits EMT, thus alleviating pulmonary and renal fibrosis in both rat pulmonary fibrosis models and mouse diabetic nephropathy models [28, 29]. Consistent with these studies, we found that the expression of E-cadherin in the cells treated with Andro increased with increasing drug concentration, while the expression of N-cadherin and Vimentin decreased. The combined therapy with Andro significantly inhibited the proliferation, migration, and invasion ability of BIU87-CisR cells.

The EMT mechanism is reversible and can be regulated directly or indirectly through various molecular mechanisms [30–32]; the activation of PI3K/AKT signaling is critical for the occurrence of EMT [33]. Phosphorylated AKT inhibits Glycogen synthase kinase 3 (GSK-3) expression, whose activity is crucial for E-cadherin expression. The results suggested that inhibiting the PI3K/AKT signaling pathway might inhibit the occurrence of EMT [34, 35]. Furthermore, studies have shown that hyperactivation of PI3K signaling is related to cancer metastasis and chemotherapy resistance [36, 37]. A study reported that the treatment of PI3K signaling pathway inhibitor NVP-BEZ235 showed marked antitumor capacities in CDDP-resistant BC cell lines [38]. Motesanib, a small-molecule inhibitor of PI3K/AKT/mTOR, enhanced the sensitivity of chemotherapy-resistant BC cells to CDDP [39]. Our study showed that PI3K-related proteins were down-regulated after Andro treatment through in vitro or in vivo experiments. The drug combination resulted in the most pronounced inhibitory effect. Currently, the evidence we have does not support the idea that Andro directly affects PI3K or other components of the PI3K/AKT pathway. To fully understand how Andro inhibits PI3K/AKT, future studies must identify the specific cellular target(s) that Andro interacts with.

In conclusion, Andro could reverse the process of EMT in CDDP-resistant BC by regulating the PI3K/AKT pathway, thus increasing the anti-tumor effect of CDDP((Fig. 8). The experimental results provided potential evidence for using Andro as a chemotherapy adjuvant in combination with Cisplatin to treat chemotherapy-resistant BC patients.

Acknowledgments

Not applicable

Funding

This work was supported by the Clinical Research Center of Jilin Provincial nephry disease and urinary system disease (urinary system tumor) clinical research center. Jilin, China. serial number: 20190905001SF

Availability of data and materials

The data generated in the present study may be requested from the corresponding author on reasonable request.

Authors' contributions

Yingjie Zhu designed the experiments and reviewed the final manuscript. Lei Xuan performed the experiments and wrote the first draft of the manuscript. Junnian Song and Ran Bi contributed to analyzed the data. All authors read and approved the manuscript.

Ethics approval and consent to participate

Animal experiments followed the guidelines of the National Institutes of Health and were authorized by the Animal Experimentation Ethics committee of the First Affiliated Hospital of Jilin University (changchun, china) (approval. No. 20210584). All methods were carried out following the relevant guidelines and regulations, and also adhered to the ARRIVE guidelines.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Andrographolide enhances the antitumor activity of cisplatin by inhibiting the PI3K/AKT signaling pathway in bladder cancer

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Abstract

Figures

Introduction

Methods

Statistical analysis

Results

Biu87-CisR cells exhibited a mesenchymal phenotype and CDDP resistance

Andro suppressed EMT in Biu87-CisR cells

Andro enhances CDDP-induced inhibition of proliferation, colony formation, and migration in Biu87-CisR cells

Andro synergistically enhances CDDP-induced apoptosis in Biu87-CisR cells

Treatment with the Andro—CDDP combination reversed the EMT process in Biu87-CisR cells

Andro—CDDP combination inhibited the PI3K/AKT pathway in Biu87-CisR cells

Effect of Andro combined with CDDP on mice bearing BIU87-CisR cell

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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