Skip to main content
NCBI home page
Cell Cycle logoLink to Cell Cycle
. 2021 Sep 14;20(21):2221–2232. doi: 10.1080/15384101.2021.1974791

Saikosaponin-A induces apoptosis of cervical cancer through mitochondria- and endoplasmic reticulum stress-dependent pathway in vitro and in vivo: involvement of PI3K/AKT signaling pathway

Jikun Du a,b,#, Daibo Song a,#, Tianshou Cao a,#, Yuanhua Li a,#, Jierong Liu a, Baohong Li a,✉,#, Li Li a,✉,#
PMCID: PMC8794532  PMID: 34520332

ABSTRACT

Cervical cancer causes considerable mortality in women worldwide. Saikosaponin-A, a triterpenoid glycoside isolated from Bupleurum falcatum, has been proven to exert anti-cancer property. In this study, we evaluated the possibility of saikosaponin-A on cervical cancer in vitro and in vivo. The results showed that saikosaponin-A induced cell death and altered cellular morphology dose-dependently. Saikosaponin-A significantly induced apoptosis in HeLa cells, confirmed by Hoechst 33,342 staining and flow cytometry. Sequentially, saikosaponin-A triggered the mitochondrial-mediated apoptosis demonstrated by deficiency of MMP, induction of Bax/Bcl-2 ratio, leakage of cytochrome c to cytoplasm, and activation of caspase-3. Moreover, ER stress also participated in the apoptosis induced by saikosaponin-A in HeLa cells as indicated by the upregulation of GPR78, CHOP and caspase-12 expression. Furthermore, HeLa cells showed increased expressions of p-PI3K and p-AKT in response to saikosaponin-A treatment. Additionally, saikosaponin-A could inhibit HeLa tumor growth in nude mice and induce apoptosis, reflected by the induction of TUNEL and the expression of cytochrome c, caspase-3 and CHOP confirmed by immunohistochemistry. These findings at least to a certain extent suggested that saikosaponin-A triggered apoptosis through both mitochondrial pathway and ER stress pathway and inhibiting PI3K/Akt signaling, thereby contributing to against cervical cancer. This work provides a new understanding of saikosaponin-A on therapeutic application in treatment of cancer, which has the potential to be a promising candidate therapeutic agent for cervical cancer patients.

KEYWORDS: Saikosaponin-a, apoptosis, cervical cancer

Introduction

As a gynecological malignant tumor, cervical cancer is the second most prevalent malignant neoplasms and the fourth primary cause of cancer mortality among women worldwide [1]. Surgery was employed as the prioritized treatment for patients with early cervical cancer, while adjuvant chemoradiotherapy was also considered and determined on the basis of the staging [2,3]. However, grievous untoward effect of chemoradiotherapy posed a certain threat to the living quality and the high cost gave rise to heavy economic burden for patients’ family [4]. Despite the current advances in diagnosis and treatment of cervical cancer, the 5-year survival rate for patients with cervical cancer in advanced-stage remained below 40% [5]. Consequently, it is important to understand the molecular pathology of cervical cancer and develop innovative strategies to cure this malignancy.

Natural products are rich sources, providing lead compounds for novel drugs development [6]. Numerous epidemiological reports have indicated that plant-derived compounds exhibited anticancer property [7–9]. Saikosaponin-A, a triterpenoid glycoside, is the major bioactive component isolated from Bupleurum falcatum, which exhibited various pharmacological activities including anti-inflammatory, antioxidant, antifibrotic, anti-epileptic, and neuromodulatory [10–14]. Recent studies have indicated that saikosaponin-A possessed strong anti-tumor properties in hepatoma cells, colon cancer cells, breast cancer cells, and lung cancer cells [15–18], which makes it a potential anti-cancer agent. However, the effect of saikosaponin-A on cervical cancer remained unclear.

To gain a further insight into the biological roles of saikosaponin-A, in this research, we attempted to investigate the effects of saikosaponin-A on cervical cancer and elucidate the possible underlying molecular mechanism both in Hela cells as an in vitro model and in a xenograft model in nude mice as an in vivo model.

Material and methods

Chemicals and reagents

Saikosaponin-A was purchased from Yuanye Biotech Company (Purity 98%, Yuanye Corp, Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco (Grand Island, USA). Penicillin-streptomycin was from Solarbio (Beijing, China). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co. LTD (Zhejiang, China). BCATM protein assay kit and Annexin V–FITC apoptosis detection kit were supplied by Beyotime (China Biotechnology). TUNEL assay kit was purchased from Keygen Biotech (Nanjing, China). Cell counting kit-8 (CCK-8) was furnished by Dojindo (Shanghai, China). Antibodies to Cytochrome c (D18C7) rabbit mAb (11,940), Bax (D2E11) rabbit mAb (5023), Bcl-2 (D17C4) rabbit mAb (3498), caspase-3 (D5R6Y) rabbit mAb (14,220), GAPDH (14C10) rabbit mAb (2118), Phospho-Akt rabbit mAb (4060), Phospho-PI3K rabbit mAb (4228) and anti-rabbit IgG alkaline phosphatase (AP)-linked antibodies were offered by Cell Signaling Technology (Danvers, MA). Antibodies to Akt rabbit mAb (ab64148) and Akt rabbit mAb (ab86714) were obtained from Abcam (Cambridge, UK).

Cell line

Human cervical cancer HeLa cells were purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 humidified incubator. HeLa cells were authenticated by short tandem repeat (STR) profiling and tested for mycoplasma contamination.

Cell viability assay

HeLa cells were planted into the 96-well plate at 5 × 104 cells/mL. After attachment, the cells were treated with different concentrations of saikosaponin-A for 24 and 48 h. Cell viability was determined using the CCK-8 assay kit according to the manufacturer’s protocol. Each experiment had three replications. Data were presented as the mean±standard deviation (SD).

Cell morphology

HeLa cells were seeded into 12-well plates (105 cells/mL) and cultivated with various concentrations of saikosaponin-A for 24 h. Then, optical microscope was used to observe morphological changes of the cells.

Hoechst 33,342 staining

HeLa cells grown in 12-well plates at a density of 105 cells/mL were treated with saikosaponin-A for 24 h. After washing triple with PBS, the cells were incubated with Hoechst 33,342 for 10 min. Then, the stained cells were observed and images were captured under a fluorescence microscopy.

Apoptosis assays

Apoptosis was detected using Annexin V-FITC/PI cell apoptosis detection kit. Following the treatment, HeLa cells were harvested and suspended in binding buffer and stained with Annexin V/PI staining solution for 10 min in the dark. The apoptosis was evaluated by flow cytometry (FACSVerse; BD Biosciences) and data were analyzed with FlowJo 7.6.1 software (Treestar, USA). Each experiment had three replications. Data were presented as the mean±SD.

Measurement of Mitochondrial Membrane Potential (MMP)

MMP was detected with fluorescent dye JC-1. After the treatment, HeLa cells were incubated in the dark with JC-1 solution at 37°C for 20 min, and rinsed with culture media. MMP were estimated by measuring the fluorescence of free JC-1 monomers (green) to JC-1 aggregates in mitochondria (red) as observed through a fluorescence microscope. Each experiment had three replications. Data were presented as the mean±SD.

Measurement of ROS

Oxidation-sensitive fluorescent probe DCFH-DA was applied to detect the intracellular ROS levels. Briefly, after the treatment, HeLa cells were washed by PBS twice, and incubated with 10 μM DCFH-DA for 30 min at 37°C in the dark. Eventually, the images of the cells were observed and photographed by a fluorescence microscope. Each experiment had three replications. Data were presented as the mean±SD.

Western blot analysis

After treated, HeLa cells were collected and lysed with RIPA lysis buffer containing PMSF (1: 100) for 30 min on ice. BCATM protein assay kit was used to measure the protein concentration. Equal amounts of proteins (25 μg) were separated on a 10% SDS-polyacrylamide gel, followed by transferred onto PVDF membrane. And then the PVDF membranes were incubated with primary antibody directed against cytochrome C, Bax, Bcl-2, Caspase 3, p-PI3K, PI3K, p-AKT and AKT at 4°C overnight followed by incubation with a horseradish peroxidase-conjugated secondary antibody for an additional 2 h at room temperature. Visualization of each band was detected by an ECL detection kit and quantified using PDQuest software (version 7.0, Bio–Rad, Hercules, CA). Each experiment had three replications. Data were presented as the mean±SD.

Animals and treatment

Female BALB/c nude mice (6–8 weeks, 20–25 g) were obtained from affiliated hospital animal center of Sun Yat-sen University (Guangzhou, China). All animal experiments were approved by the Institutional Animal Care and Use Committee at Guangdong Medical University and carried out in compliance with the guidelines. The right flanks of nude mice were injected with HeLa cells in the logarithmic growth phase to set up cervical cancer xenograft model (1 × 106 cells resuspended in 0.1 mL of PBS). The tumor growth and body weight of the mice were monitored weekly. Tumor volumes were calculated as the formula: Tumor volume (mm3) = (length × width2)/2. When tumors reached around a size of 100 mm3 the models were established. Subsequently, tumor-bearing mice were randomly assigned to two equal groups (n = 6) for intragastric administration of either saikosaponin-A (15 mg/kg) or the vehicle (i.g.) respectively every other day for 4 weeks. Tumor volume was monitored by calipers twice weekly. Each tumor xenograft was excised and weighed when nude mice were sacrificed at the end of the experiment. Parts of tumor tissues were fixed in formaldehyde solution rapidly, and were wax embedded for histopathological and immunohistochemical analysis.

H&E staining

The wax embedded tumor sample was cut into 5 μm thick section, and stained with hematoxyline-eosin (H&E). The stained tissue sample was examined under a light microscope.

Immunohistochemically analysis

The thickness sections (5 μm) cut from tumor tissues were mounted on the glass slides, then deparaffinized, quenched and incubated with the primary antibodies against to caspase-3, CHOP and caspase-12, respectively. After that, the slides were blocked with goat serum for 20 min and then incubated with the secondary antibody at 37°C, respectively. The final positive staining was counterstained by diaminobenzidine for 10 min at room temperature. Typical images were observed under a light microscope.

TUNEL staining

The wax embedded tumor samples were sliced into 5 μm. Apoptotic cells in tumor tissue were determined by the TUNEL assay kit according to the manufacturer’s instruction. Apoptotic cells were observed and photographed under microscope.

Statistical analysis

All the results are presented as the means±SD of triplicate experiments. Statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). Differences between groups were performed using Student’s t-test (2-group comparisons) or one-way analysis of variance followed by Tukey’s post hoc test (>2 groups). P < 0.05 was considered to indicate a statistically significant difference.

Results

Saikosaponin-A induced the growth inhibition and cellular morphology change in HeLa cells

Firstly, CCK-8 assay was performed to evaluate the cytotoxicity of saikosaponin-A on HeLa cells. Saikosaponin-A at concentrations of 5, 10, and 15 μM observably reduced cell viability to 93.08 ± 2.44%, 69.54 ± 5.03%, and 42.47 ± 2.50% of control in HeLa cells at 24 h. 20 μM saikosaponin-A caused massive cell death in HeLa (Figure 1b). Therefore, saikosaponin-A at a concentration of 15 μM was selected in the subsequent tests. The antigrowth effect of saikosaponin-A was further confirmed by cellular morphology changes. Untreated HeLa cells were tightly attached to cell plates distinguished by a well-organized monolayer with a regular polygonal appearance. Whereas prominent changes of cellular shape were found after saikosaponin-A administration, such as disconnected from each other and broken cell membrane. Even high doses of saikosaponin-A could lead HeLa cells falling off from the cell plates and floated in the culture medium (Figure 3a). These results demonstrated that saikosaponin-A could induce cell death in HeLa cells.

Figure 1.

Figure 1.

Open in a new tab

Saikosaponin-A inhibits the growth of HeLa cells. (a) Chemical structure of saikosaponin-A Chemical formula: C42H68O13, molecular weight: 781 g/mol, from pubchem https://pubchem.ncbi.nlm.nih.gov/ compound/167,928 (b) Effect of saikosaponin-A on the proliferation in HeLa cells. HeLa cells were exposed to different concentrations of saikosaponin-A for 24 h and 48 h. After that the cell proliferation was determined by CCK–8 assay. The data are expressed as the means ± SD from three independent experiments. *indicates significant difference (p < 0.05)

Figure 3.

Figure 3.

Open in a new tab

Saikosaponin-A induced morphology change, intracellular ROS generation and the loss of MMP in HeLa cells. HeLa cells were exposed to different concentrations of saikosaponin-A for 24 h. (a) The morphological features were observed under microscope. After that the cells were incubation with the fluorescent probe DCFH-DA for 30 min, ROS generation was detected using a fluorescence microscope. The reductions of MMP were determined by JC-1 probe and fluorescence microscope. Red fluorescence indicates normal mitochondria and green fluorescence indicates damaged mitochondria. (b) The graph shows the ROS fluorescence quantitative analysis. (c) The graph shows the red/green fluorescence intensity ratio quantitative analysis

Saikosaponin-A triggered apoptosis in HeLa cells

Subsequently, whether the anticancer effect of saikosaponin-A against HeLa cells was associated with apoptosis was assessed. According to Hoechst 33,342 staining, untreated HeLa cells remained uniform blue stained, with a single round spots in the nuclear. Small intact fragments termed apoptotic bodies, chromatin condensation, plasma membrane blebbing, and shrinkage was observed in saikosaponin-A treated cells. Furthermore, the apoptosis triggered by saikosaponin-A in HeLa cells was ascertained by flow cytometry. After saikosaponin-A administration, the percentages of apoptotic cells were increased to 6.96 ± 0.30% (5 μM), 18.32 ± 0.82% (10 μM) and 48.80 ± 2.48% (15 μM) (Figure 2b). These results suggested that saikosaponin-A could induce apoptosis in HeLa cells dose-dependently.

Figure 2.

Figure 2.

Open in a new tab

Saikosaponin-A triggers apoptosis in HeLa cells. HeLa cells were exposed to different concentrations of saikosaponin-A for 24 h. (a) Apoptotic condensed nuclear changes were determined via Hoechst 33,342 staining and conduct by a fluorescence microscopy. (b) (c) Cell apoptosis was analysis by Annexin V–FITC/PI staining and flow cytometry

Saikosaponin-A caused mitochondrial dysfunction and activated apoptosis through of the intrinsic mitochondrial signaling pathway in HeLa cells

JC-1, a cationic dye, was applied to detect the MMP of HeLa cells following exposure to saikosaponin-A. Sure enough, compared with the control group, saikosaponin-A remarkably triggered the loss of MMP in HeLa cells at 24 h (Figure 3). Then, the intracellular ROS formation was conducted by fluorescent probe DCFH-DA. Figure 3 showed that an enhancement of ROS formation was observed in HeLa cells challenged with saikosaponin-A. Hence, these results suggested that saikosaponin-A could dramatically decrease MMP and increase ROS formation in HeLa cells. Then, the expressions of apoptosis-associated proteins cytochrome c, caspase-3, Bax and Bcl-2 were evaluated. Western blot analysis revealed that the release of cytochrome c was observed in HeLa cells incubated in saikosaponin-A. Meanwhile, saikosaponin-A effectively increased the expression of pro-apoptotic proteins Bax, while simultaneously decreased the level of anti-apoptotic Bcl-2 in HeLa cells. Furthermore, capsase-3, an executor of apoptosis, was upregulated in HeLa cells treated with saikosaponin-A (Figure 4). These results indicated that saikosaponin-A could efficiently induce apoptosis through mitochondrial apoptotic pathway.

Figure 4.

Figure 4.

Open in a new tab

Saikosaponin-A activates the mitochondrion-mediated apoptotic pathway in HeLa cells. Effect of saikosaponin-A on the release of cytochrome c and the expression of Bax, Bcl-2 and caspase-3 in HeLa cells. HeLa cells were exposed to different concentrations of saikosaponin-A for 24 h. The release of cytochrome c, Bax, Bcl-2 and caspase-3 was confirmed by western blot. GAPDH was used as loading control

Saikosaponin-A induced ER stress dependent apoptosis

The ER stress-specific protein marker GRP78, CHOP and caspase 12 were detected by western blot to investigate the influence of saikosaponin-A on the role of ER stress in the process of apoptosis in HeLa cells. Figure 5 showed that the levels of GRP78, CHOP and caspase 12 were augmented in HeLa cells administrated with saikosaponin-A. These results implied that ER stress played a crucial role in the saikosaponin-A-induced apoptosis in cervical cancer cell.

Figure 5.

Figure 5.

Open in a new tab

Saikosaponin-A activates the ER stress apoptotic pathway in HeLa cells. Effect of saikosaponin-A on the the expression of GPR78, CHOP and caspase-12 in HeLa cells. HeLa cells were exposed to different concentrations of saikosaponin-A for 24 h. The release of the expression of GPR78, CHOP and caspase-12 was confirmed by western blot. GAPDH was used as loading control

Saikosaponin-A inactivated PI3K/AKT signaling in hela cells

Subsequently, we investigated the impact of saikosaponin-A on PI3K/AKT signaling. Saikosaponin-A treatment markedly decreased the ratio of p/t-PI3K and p/t-AKT, which indicated that saikosaponin-A could restrain the PI3K/AKT signaling in HeLa cells (Figure 6a). Furthermore, LY294002 was applied to specify the role of PI3K/AKT signaling in cellular fate. LY294002 and saikosaponin-A reduced the viability and the apoptotic rate of HeLa cells (Figure 6b, c), which were strongly correlated with the reduced phosphorylation of PI3K and AKT (Figure 6a). There results at least in part suggested that saikosaponin-A might exert its induction of apoptosis through PI3K/AKT signaling pathway.

Figure 6.

Figure 6.

Open in a new tab

Saikosaponin-A suppression regulated PI3K/AKT signaling in HeLa cells. (a) Effect of saikosaponin-A on PI3K/AKT pathway in HeLa cells. HeLa cells were exposed to saikosaponin-A and LY294002 for 24 h. Cell extracts were analyzed by Western blot with antibodies. (b) Effect of saikosaponin-A and LY294002 on the apoptotic rate in HeLa cells. (c) Effect of saikosaponin-A and LY294002 on the cell viability in HeLa cells

Saikosaponin-A inhibited tumor growth in vivo

Finally, to estimate the anti-tumor effect of saikosaponin-A in vivo, HeLa tumor bearing xenograft was constructed. Saikosaponin-A disposed at 15 mg/kg was very well tolerated by nude mice without obvious toxicity throughout the course of study. Remarkably, tumors in the saikosaponin-A group developed slower than the vehicle groups (Figure 7a). Additionally, compared with the vehicle group, an enhancement in cell death in the tumors of saikosaponin-A groups was confirmed by H&E staining (Figure 7b). Furthermore, the level of TUNEL, as the cellular apoptosis marker, was increased tremendously in saikosaponin-A-treated groups compared with the vehicle group, which suggested saikosaponin-A induced apoptosis in HeLa xenografts. Simultaneously, cytochrome c, caspase 3, and CHOP expression were dramatically increased in saikosaponin-A administration group confirmed by immunohistochemistry, which demonstrated that saikosaponin-A could augment the antitumor property in HeLa xenografts was associated with its induction of mitochondrial – and ER stress-dependent apoptosis. These results provide strong evidence for saikosaponin-A on suppression tumor growth by induction of apoptosis in vivo.

Figure 7.

Figure 7.

Open in a new tab

Saikosaponin-A inhibited HeLa tumor growth of a xenograft model in nude mice by inducing apoptosis. (a) Effects of saikosaponin-A on the tumor volume of a xenograft model in nude mice. The nude mice were subcutaneously inoculated with HeLa cells on the right flank. Mice were administrated with saikosaponin-A for 4 weeks and the tumor volumes were monitored twice weekly. The mice were sacrificed after 4 weeks’ treatment and the tumors were isolated and tumors size were caculated. (b) Saikosaponin-A induces apoptosis in vivo. Histopathological changes in the tumor tissue were evaluated by HE staining. The expression of caspase-3, CHOP and cytochrome c in xenograft tumor tissues was observed by immunohistochemical staining. Immunohistochemical staining of TUNEL in HeLa cell xenografts of nude mice showed that saikosaponin-A treatment induced cell apoptosis in vivo.

Discussion

Natural products, exhibiting fewer side effects than chemical synthetic drugs in the treatment of various diseases, have been attracting increasing attention in the world [19]. In vivo and in vitro studies manifested that saikosaponin-A exhibited pharmacologic activities against a variety of diseases such as hyperlipidemia [20], hepatic injury [21], digestive ulcers [22], and inflammation [10]. Moreover, increasing evidences indicated that saikosaponin-A possessed potential anti-cancer activity. Saikosaponin-A inhibited breast cancer growth and metastasis via downregulation of CXCR4 [23] and shifting Th1/Th2 balance [17]. Additionally, saikosaponin-A could induce DNA damage and caspase activation through caspase-4 in colon carcinoma cells [15]. However, there is no evidence on the effect of saikosaponin-A against cervical cancer. Here, we used HeLa cells as an in vitro model and a HeLa xenografted nude mice as an in vivo model, to investigate the potential effect of saikosaponin-A on cervical cancer. Our data illustrated that saikosaponin-A triggered apoptosis through both mitochondrial- and ER stress-dependent pathway in vivo and in vitro. These inductions appeared to correlate with the suppression of PI3K/AKT signaling pathway.

As an essential programmed cell death, apoptosis plays a fateful role in homeostasis of multicellular organisms. Evasion of apoptosis, resulting in uncontrolled growth, may be a hallmark for all types of cancer [24]. Therefore, compounds reviving apoptosis of cancer cell may be promising candidates to cure cancer [25]. In the present study, we found that saikosaponin-A could restrain cellular growth and trigger apoptosis in HeLa cells. Up to date, induction of apoptosis is conducted through death receptors-mediated pathway (extrinsic) and mitochondrial-dependent pathway (intrinsic) [26]. The mechanism of mitochondria-mediated apoptosis mainly depends on mitochondria dysfunction, which resulted in impaired mitochondrial electron transport chain, loss of MMP, and ROS production [27,28]. The decrease of MMP implies that the cell is in the early stages of apoptosis. Impaired mitochondria generate more ROS and further accelerate apoptosis [29]. Bcl-2 family proteins, including the anti-apoptotic proteins Bcl-2 as well as the pro-apoptotic proteins Bax, are profoundly responsible for mitochondrial apoptotic pathway [30]. The balance of Bax and Bcl-2 is a crucial determinant for cellular fate. A high ratio of Bax/Bcl-2 caused collapse of MMP, ROS generation and release of cytochrome c into cytosol, which activates caspase-9 and then triggers the apoptotic executor caspase–3 [31,32]. Equol could induce mitochondria-dependent apoptosis in human gastric cancer MGC-803 cells [33]. Oridonin induced cell cycle arrest and mitochondrial-mediated apoptosis in esophageal cancer cells [34]. It’s reported that saikosaponin-A could induce apoptosis through mitochondria-dependent pathway in hepatic stellate cell line HSC-T6 [35]. In this investigation, remarkable collapse of MMP and induction of ROS formation were found in saikosaponin-A treated HeLa cells. Saikosaponin administration increased the ratio of Bax/Bcl-2, followed by the releasing of cytochrome c to the cytoplasm and upregulation of cleaved caspase-3 in HeLa cells. These findings confirmed the involvement of the mitochondrial pathway in saikosaponin-A-triggered apoptosis.

Endoplasmic reticulum (ER) is characterized as a vital subcellular organelle participating in the synthesis, modification, and sorting of protein in eukaryotic cells [36]. Previous studies identified that unfolded proteins accumulated in ER lumen ultimately resulting in ER stress, which act as a defense mechanism to improve the survival of cells through the unfolded protein response (UPR) to establish ER homeostasis by halting protein synthesis and bolstering protein folding capacity. Once the UPR is triggered, GRP78, a marker for ER stress, dissociates from ER transmembrane receptors. If the ER stress is too aggravated or persisted, the UPR fails to reestablish cellular homeostasis, and ER stress switches to initiate apoptosis through several signaling cascades, such as caspase-12 and CHOP [37]. Therefore, compounds that cause excessive ER stress can be an effective strategy to eliminate cancer cells [38]. Corosolic acid might serve as a candidate in the treatment of human castration resistant prostate cancer through inducing ER stress-dependent apoptosis via activation of IRE-1/JNK, PERK/CHOP and TRIB3 [39]. Aloe-Emodin reveals its anti-tumor ability in the treatment of colorectal cancer through inducing ER stress-dependent apoptosis [40]. However, the influence of saikosaponin-A on apoptosis involving ER stress remains largely unsolved. In this study, we have for the first time shown saikosaponin-A triggered ER stress in the apoptotic process of HeLa cells. Nevertheless, further studies are extensively needed to define the exact role of saikosaponin-A in ER stress mediated apoptosis in HeLa cells.

Similar results were found in cervical cancer nude mice model. Saikosaponin-A administration could decrease tumor growth of HeLa cells xenografted mice without causing mortality and markedly strengthened apoptosis as shown by immunostaining in the tumors, which was associated with its induction of mitochondrial- and ER stress-dependent apoptosis. This phenomenon reflects the potential clinical importance of saikosaponin-A on cervical cancer therapy. However, more experiments are needed to determine the safety of saikosaponin-A in human body for clinical application.

Generally, an in-depth understanding of signal transduction may provide new targets for oncotherapy. PI3K/Akt signaling is a well-known and crucial signaling referring to manipulate proliferation, transformation, growth, apoptosis, drug resistance and other courses in multifarious types of cancers [41]. Scopoletin may prove beneficial in the management of cervical cancer by triggering apoptosis and inactivation of PI3K/Akt signaling pathway [42]. Fucosterol exerts anticancer effects on human cervical cancer cell lines by downregulation of PI3K/Akt signaling pathway [43]. Therefore, PI3K/AKT pathway becomes a potential target for cervical cancer prevention and treatment. In this study, saikosaponin-A could inhibit HeLa cells growth via inhibition of PI3K/Akt pathway. Nevertheless, the full molecular milieu linking all these events needs further study.

In this study, we presented new insights on the potential implication of PI3K/AKT in mitochondria- and ER stress-dependent apoptosis mediated by saikosaponin-A (Figure 8). However, the mechanisms of saikosaponin-A mediating its therapeutic effects against cervical cancer remain largely unsolved. Attempts to unravel the complete signaling molecules linking all these events will pave the way for developing preventive and/or therapeutic strategies of cervical cancer. Taken together, saikosaponin-A might be a promising therapeutic strategy to combat cervical cancer deserving further research.

Figure 8.

Figure 8.

Open in a new tab

Illustration representing the mechanisms of saikosaponin-A-induced apoptosis in cervical cancer

Acknowledgments

This work was supported by the Guangdong Natural Science Foundation (No. 2020A1515010840), Medical Scientific Research Foundation of Guangdong Province (A2021224), the Project of Educational Commission of Guangdong Province (2020KTSCX047 & 4SG20124G & 4SG21207G), and Fund of Guangdong Medical University (GDMUZ2020005).

Correction Statement

This article has been republished with minor changes. These changes do not impact the academic content of the article.

Funding Statement

This work was supported by the project of educational commission of guangdong province [2020KTSCX047 & 4SG20124G & 4SG21207G]; Fund of Guangdong Medical University [GDMUZ2020005]; medical scientific research foundation of guangdong province [A2021224]; Guangdong Province Universities and Colleges Pearl River Scholar Fund [4SG21006G].

Author statement

Li Li and Baohong Li generated the idea. Li Li designed the project. Daibo Song, Yuanhua Li and Li Li performed the experiments. Li Li, Tianshou Cao and Jierong Chen analyzed the data. Li Li and Jikun Du wrote the manuscript.

Ethical statement

This work does not contain any studies with human participants or animals performed by any of the authors.

Disclosure statement

No potential conflict of interest was reported by the author(s).

References

  • [1].Lai TO, Boon SS, Law PT, et al. Oncogenicitiy comparison of human papillomavirus type 52 E6 variants. J Gen Virol. 2019;100(3):484–496. [DOI] [PubMed] [Google Scholar]
  • [2].Pfaendler KS, Tewari KS.. Changing paradigms in the systemic treatment of advanced cervical cancer. Am J Obstet Gynecol. 2016;214(1):22–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Tangjitgamol S, Katanyoo K, Laopaiboon M, et al. Adjuvant chemotherapy after concurrent chemoradiation for locally advanced cervical cancer. Cochrane Database Syst Rev. 2014;12:CD010401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Lee MY, Shen MR.. Epithelial-mesenchymal transition in cervical carcinoma. Am J Transl Res. 2012;4:1–13. [PMC free article] [PubMed] [Google Scholar]
  • [5].Sankaranarayanan R, Swaminathan R, Brenner H, et al. Cancer survival in Africa, Asia, and Central America: a population-based study. Lancet Oncol. 2010;11(2):165–173. [DOI] [PubMed] [Google Scholar]
  • [6].Harvey AL, Edrada-Ebel R, Quinn RJ. The re-emergence of natural products for drug discovery in the genomics era. Nat Rev Drug Discov. 2015;14:111–129. [DOI] [PubMed] [Google Scholar]
  • [7].Thazin A, Zhipeng Q, Kortschak R, et al. Understanding the effectiveness of natural compound mixtures in cancer through their molecular mode of action. Int J Mol Med Sci. 2017;18:656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Telang NT, Nair HB, Wong GYC. Growth inhibitory efficacy of Cornus officinalis in a cell culture model for triple-negative breast cancer. Oncol Lett. 2019;17:5261–5266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Li X, Wang W, Fan Y, et al. Anticancer efficiency of cycloartane triterpenoid derivatives isolated from Cimicifuga yunnanensis Hsiao on triple-negative breast cancer cells. Cancer Manage Res. 2018;10:6715–6729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Zhu J, Luo C, Wang P, et al. aikosaponin A mediates the inflammatory response by inhibiting the MAPK and NF-kappaB pathways in LPS-stimulated RAW 264.7 cells. Exp Ther Med. 2013;5(5):1345–1350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Fu Y, Hu X, Cao Y, et al. Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts. Free Radic Biol Med. 2015;89:777–785. [DOI] [PubMed] [Google Scholar]
  • [12].Wu SJ, Tam KW, Tsai YH, et al. Curcumin and saikosaponin a inhibit chemical-induced liver inflammation and fibrosis in rats. Am J Chin Med. 2010;38(1):99–111. [DOI] [PubMed] [Google Scholar]
  • [13].Ye M, Bi YF, Ding L, et al. Saikosaponin a functions as anti-epileptic effect in pentylenetetrazol induced rats through inhibiting mTOR signaling pathway. Biomed Pharmacother. 2016;81:281–287. [DOI] [PubMed] [Google Scholar]
  • [14].Mao X, Miao G, Tao X, et al. Saikosaponin a protects TBI rats after controlled cortical impact and the underlying mechanism. Am J Transl Res. 2016;8:133–141. [PMC free article] [PubMed] [Google Scholar]
  • [15].Kang SJ, Lee YJ, Kang SG, et al. Caspase-4 is essential for saikosaponin a-induced apoptosis acting upstream of caspase-2 and γ-H2AX in colon cancer cells. Oncotarget. 2017;8(59):100433–100448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Zhang CY, Jiang ZM, Ma XF, et al. Saikosaponin-d inhibits the hepatoma cells and enhances chemosensitivity through SENP5-dependent inhibition of Gli1 SUMOylation under hypoxia. Front Pharmacol. 2019;10:1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Zhao X, Liu J, Ge S, et al. Saikosaponin A inhibits breast cancer by regulating Th1/Th2 Balance. Front Pharmacol. 2019;10:624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Tang JC, Long F, Zhao J, et al. The effects and mechanisms by which saikosaponin-d enhances the sensitivity of human non-small cell lung cancer cells to gefitinib. J Cancer. 2019;10(26):6666–6672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Hur SJ, Kang SH, Jung HS, et al. Review of natural products actions on cytokines in inflammatory bowel disease. Nutr Res. 2012;32(11):801–816. [DOI] [PubMed] [Google Scholar]
  • [20].Feng P, Xu Y, Tong B, et al. Saikosaponin a attenuates hyperlipidemic pancreatitis in rats via the PPAR-gamma/NF-kappaB signaling pathway. Exp Ther Med. 2020;19:1203–1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Li X, Li X, Lu J, et al. Saikosaponins induced hepatotoxicity in mice via lipid metabolism dysregulation and oxidative stress: a proteomic study. BMC Complement Altern Med. 2017;17(1):219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Zhou F, Wang N, Yang L, et al. Saikosaponin A protects against dextran sulfate sodium-induced colitis in mice. Int Immunopharmacol. 2019;72:454–458. [DOI] [PubMed] [Google Scholar]
  • [23].Wang Y, Zhao L, Han X, et al. Saikosaponin A inhibits triple-negative breast cancer growth and metastasis through downregulation of CXCR4. Front Oncol. 2020;9:1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Carneiro BA, El-Deiry WS . Targeting apoptosis in cancer therapy. Nat Rev Clin Oncol.2020; 17(7):395–417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–674. [DOI] [PubMed] [Google Scholar]
  • [26].Kim H-S. Insulin-like growth factor-binding protein 3 induces caspase-dependent apoptosis through a death receptor-mediated pathway in MCF-7 human breast cancer cells. Cancer Res. 2004;64(6):2229–2237. [DOI] [PubMed] [Google Scholar]
  • [27].Susin SA, Zamzami N, Kroemer G. The cell biology of apoptosis: evidence for the implication of mitochondria. Apoptosis. 1996;1(4):231–242. [Google Scholar]
  • [28].Komal P, Anand KT. Mitochondrial dynamics, a key executioner in neurodegenerative diseases. Mitochondrion. 2019;47:151–173. [DOI] [PubMed] [Google Scholar]
  • [29].Marchetti P, Castedo M, Susin SA, et al. Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med. 1996;1(3):1155–1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Aouacheria A, Cibiel A, Guillemin Y, et al. Modulating mitochondria-mediated apoptotic cell death through targeting of Bcl-2 family proteins. Recent Pat DNA Gene Seq. 2007;1(1):43–61 [DOI] [PubMed] [Google Scholar]
  • [31].Armstrong JS. Mitochondria: a target for cancer therapy. Br J Pharmacol. 2006;147(3):239–248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Armstrong JS. The role of the mitochondrial permeability transition in cell death. Mitochondrion. 2006;6(5):225–234. [DOI] [PubMed] [Google Scholar]
  • [33].Yang Z, Zhao Y, Yao Y, et al. equol induces mitochondria-dependent apoptosis in human gastric cancer cells via the sustained activation of ERK1/2 pathway. Mol Cells. 2016;39(10):742–749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Jiang JH, Pi J, Jin H, et al. Oridonin-induced mitochondria-dependent apoptosis in esophageal cancer cells by inhibiting PI3K/AKT/mTOR and Ras/Raf pathways. J Cell Biochem. 2019;120(3):3736–3746. [DOI] [PubMed] [Google Scholar]
  • [35].Chen CH, Chen MF, Huang SJ, et al. Saikosaponin A induces apoptosis through mitochondria-dependent pathway in hepatic stellate cells. Am J Chin Med. 2017;45(2):1–18. [DOI] [PubMed] [Google Scholar]
  • [36].Almanza A, Carlesso A, Chintha C, et al. Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications. FEBS J. 2019;286:241–278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [37].Oakes SA. Endoplasmic reticulum stress signaling in cancer cells. Am J Pathol. 2020;190(5):934–946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Oakes SA. Endoplasmic reticulum proteostasis: a key checkpoint in cancer. Physiol Cell Physiol. 2017;312(2):C93–c102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Ma B, Zhang H, Wang Y, et al. Corosolic acid, a natural triterpenoid, induces ER stress-dependent apoptosis in human castration resistant prostate cancer cells via activation of IRE-1/JNK, PERK/CHOP and TRIB3. J Exp Clin Cancer Res. 2018;37(1):210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Cheng C, Dong W. Aloe-emodin induces endoplasmic reticulum stress-dependent apoptosis in colorectal cancer cells. Med Sci Monit. 2018;24:6331–6339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [41].Hennessy BT, Smith DL, Ram PT, et al. Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov. 2005;4(12):988–1004. [DOI] [PubMed] [Google Scholar]
  • [42].Tian Q, Wang L, Sun X, et al. Scopoletin exerts anticancer effects on human cervical cancer cell lines by triggering apoptosis, cell cycle arrest, inhibition of cell invasion and PI3K/AKT signalling pathway. J BUON. 2019;24:997–1002. [PubMed] [Google Scholar]
  • [43].Jiang H, Li J, Chen A, et al. Fucosterol exhibits selective antitumor anticancer activity against HeLa human cervical cell line by inducing mitochondrial mediated apoptosis, cell cycle migration inhibition and downregulation of m-TOR/PI3K/Akt signalling pathway. Oncol Lett. 2018;15:3458–3463. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]

Articles from Cell Cycle are provided here courtesy of Taylor & Francis

RESOURCES