Introduction
Breast cancer ranks the first in the incidence and mortality of malignant tumors in Chinese women, and the incidence is still rising [
1,
2]. Breast cancer has undoubtedly become a major threat to women’s health globally. Triple negative breast cancer (TNBC) is a special molecular subtype pattern which is characteristic of negative for estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2). According to the American Cancer Society, nearly 260,000 women are diagnosed with breast cancer each year, of which 15–20% are TNBC. A larger proportion of younger women is reported with TNBC than older patients [
3,
4]. The incidence of breast cancer in young women has increased worldwide in recent years [
5]. In the United States, about 11% of breast cancer patients are between the age of 35 and 45 years, and in Asia are 9.5–12%. TNBC is the most aggressive breast cancer subtype in young patients. Compared with other breast cancer subtypes, patients with TNBC had a higher mortality, worse overall survival and higher recurrence rate. Unlike other molecular subtypes of breast cancer, patients with TNBC do not benefit from hormonotherapy and HER-2 targeted therapy. The limited choices of medical treatment for them are usually chemotherapy and immunotherapy but with limited therapeutic effect. Therefore, there is still a tremendous unmet need for the development of novel therapeutics for TNBC [
6].
CAXII inhibitor, as a potential cancer treating agent, has been found to impede the creation and transportation of bicarbonate ions into the cells through anion exchangers and Na+/HCO3 − co-transporters, consequently regulating intracellular pH and extracellular acidosis that affect cancer development in the tumor micro-environment [
7,
8]. CAXII inhibitor has been thought to improve the apoptosis of cancer cells, but related mechanisms still need to be further explored [
9]. In addition, selectively silencing of
CAXII gene significantly inhibites the migration and invasion of TNBC cells, which ensures the potential of CAXII inhibitor in TNBC treatment [
10].
Tribulus terrestris L. (TT), a plant in many regions of Asia and Africa, has been used in traditional Chinese medicine as an herb to treat breast diseases for thousands of years by the formation of oral administration decoction [
11]. Nowadays, evidence has proven that the extracts of TT has many medical effects such as antihypertension, diuretic effect and cancer suppression [
12]. TT extracts have been found to regulate the apoptosis and metastasis of cancer cells by regulating NF-κB signaling [
11].
Tiliroside (
TS, C
30H
26O
13), as a major active ingredient of TT, has been further found to possess anti-inflammatory, anticholinesterase and antioxidant activities [
13]. It has been reported to suppress cancer development by regulating NF-κB signaling pathway or MAPK/JNK/p38 axis in leukemia cells [
14]. Recently, we further uncovered its anticancer effects on Hepatocellular carcinoma (HCC) cells by targeting CAXII as a novel CAXII inhibitor [
9]. One scientific question we asked was whether or not
TS had the similar anticancer effect against breast cancer.
Thus, the purposes of this study were to investigate the association between CAXII expression level and survival probability of TNBC patients, to examine the effects of TS on TNBC cells and to explore the related mechanisms in vitro and in vivo.
Materials and methods
Gene profile overview and survival analysis
Publicly accessed datasets were used to evaluate the prognostic values of CAXII in TNBC and differentiated expression of CAXII. Gene profile overview were conducted in GENTS platform (
http://gent2.appex.kr/gent2/) and PEGIA platform (
http://gepia.cancer-pku.cn). The Kaplan–Meier survival curves of CAXII in TNBC were generated based on the datasets of triple negative breast cancer RNA-seq using Kaplan–Meier plotter (
http://kmplot.com/analysis), in which the optimal cutoff value for CAXII expression was used to classify the patients into two groups, high vs. low CAXII expression.
Cell culture and transduction of luciferase lentivirus
Human triple-negative breast cancer cell MDA-MB-231, BT-549, and human breast normal epithelial cell MCF-10 A were purchased from American Type Culture Collection (ATCC, USA) and were characterized using STR (Short Tandem Repeat) analysis for identity verification of human cell lines in January, 2022. The cells were cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC, USA), RPMI1640 (ATCC, USA) or Leibovitz’s L-15 medium (LLM, ATCC, USA), respectively, with 10% fetal bovine serum (ATCC, USA), or Mammary Epithelial Cell Growth (MEGM, USA) completed with MEGM bullet kit (Lonza, USA). Cells were cultured in an incubator under the condition of humidified incubator with 5% CO2 at 37 °C.
Both MDA-MB-231 and BT-549 cells were transfected with CMV-Firefly luciferase-IRES-Puro lentivirus (Cellomics,USA), and characterized by STR analysis as described previously [
15].
CAXII gene knockout
For establishing CAXII knockout clones, as described in our previous study [
9], MDA-MB-231 and BT-549 were both transfected with 1 µg of CAXII CRISPR guide RNA vector plus vector coding for puromycin resistance by applying Metafectene Pro (Biontex, Munich, Germany), respectively. For non-KO clones (as a mock group), CAXII CRISPR guide RNA vector has not been applied. Cells were then selected by puromycin for 16 h. CAXII expression was confirmed after 24 h by RT-qPCR.
RNA extraction and quantitative RT-PCR
Total RNA was extracted from MDA-MB-231, BT-549 and MCF-10 A cells by using the RNeasy mini kit (Qiagen, Germany) [
9]. The concentration and purity of total RNA were determiend by using an Epoch microplate spectrophotometer (Biotek, USA). Total RNA was reverse-transcribed to cDNA templates by using an AffinityScript multi temperature cDNA synthesis kit (Agilent technologies, CA, USA). The expression of detected genes was determined using the SYBR Green PCR Kit (Qiagen, Germany) on a 7500 Fast Real-time PCR System (Life Technologies, USA). All the primer sequences used in this study and qPCR reaction conditions were described in our previous study[
9]. Each sample was analyzed in triplicate. After each PCR amplification, dissociation curve was assessed. The relative expression levels of genes were expressed as a fold change relative to
GAPDH using the 2
−ΔΔCt method.
CAXII activity assessment
CAXII activity inhibition assay was performed following the instructions as described in our previous study [
9]. For details, the
TS was purchased from SIGMA-ALDRICH (USA), the enzyme activity was assessed by monitoring 4-nitrophenol (4-NP/pNP) (SIGMA-ALDRICH, USA). Absorbance of the spontaneous hydrolysis of the substrate alone and substrate treated with
TS was subtracted from esterase activity in absence and presence of
TS. In addition, we added potent CAXII inhibitor (U-104) (EMD Millipore corporation, USA) as a positive control. Plotting absorbance (Y-axis) and time (min) (X-axis) was used to evaluate the slope of the initial rate of enzyme activity. The percentage of enzyme activity was then calculated. By applying the non-linear least squares method, the IC50 values were finally calculated.
Caspase-3 activity
For conducting the CaspACE™ Assay, 2 × 10
6 MDA-MB-231(or BT-549) cells cultured in 2 mL medium were treated with either 40 µM
TS or same amount of DMSO as the induced apoptosis group (72 h after intervention), and 3 µL Z-VAD-FMK inhibitor was added in 72 h intervention cells as the inhibited apoptosis groups. The mock groups were a normal control (NC). The plate was incubated at 37 °C in a humidified incubator with 5% CO
2 for 16 h. After the centrifugation of cell lysates, the cell supernatant fractions were harvested for CASP3 activity measurement. In addition, the protein concentration of each sample was determined by the BCA protein assay (Thermo Fisher Scientific, USA), and the pNA Calibration Curves were made by a colorimetric assay system. CASP3 Specific Activity (SA) was calculated as the formulas as described elsewhere [
9,
15].
Detection of intracellular pH (pHi), extracellular pH (pHe) and lactate level
Intracellular pH was evaluated using the fluorometric intracellular pH assay kit (Sigma, Germany, MAK-150) as previously described [
16]. Fluorescent BCFL-AM indicator was used to measure pHi fluctuations in the fluorometric intracellular pH kit for the cells treated by various conditions. The cells were seeded in black-wall plates with 6 × 10
4 cells in each well, and were incubated for 24 h. The medium was replaced with BCFL-AM reagent prepared in 100 mL of HBS solution (Hank’s buffer with 20 mM HEPES, 5 mM probenecid), then the cells were incubated at 37 °C in an atmosphere of 5% CO2 for 30 min (with avoiding light).
TS compound at the doses of 0, 20, 40 and 80 µM was added to the HBS solution. After 5 min incubation, the measurement was performed at wavelengths of 490 nm (excitation) and 535 nm (emission) in spectrofluorometry (Spectramax, M5). Extracellular pH (pHe) and lactate levels were evaluated directly using a commercial kit on the blood gas device (ABL90 FLEX PLUS, Radiometer, Copenhagen, Denmark).
MTS cell proliferation assay
Cell proliferation MTS assay was performed by adding 20 µL of MTS solution (Promega, USA) into each well in a dark hood at different incubation time points (24 h, 48 h, 72 h, 96 h) following the manufacturer’s instruction. Microplate Spectrophotometer (Biotek, USA) was used to detect the cells’ absorbance at the wavelength of 450 nm. Triplicate were conducted for each condition at each time point. The proliferation inhibition rate was calculated based on the formula: inhibition rate = (1-Absorbance of treated sample /Absorbance of control sample)×100%.
Wound healing assay
MDA-MB-231 and BT-549 cells were cultured in 6-well tissue culture plates with approximately 1 × 10
6 cells in each well, respectively. Single wound was scratched in each plate when cell monolayer grew to nealry 90% confluence. 40 µM
TS in DMSO or the same final concentration of DMSO were added for different groups. Images were taken at 0 h, 24 and 48 h of incubation after the wound, respectively, for the average of wound closure measurement [
17]. The wound boundaries were defined by applying ImageJ (version 1.52a; National Institutes of Health, USA) as described in our previous study [
15].
Transwell invasion assay
The MDA-MB-231 and BT-549 cells (1 × 104) were seeded in each upper layer of culture insert of 3.0 µM pore size transell chamber (Falcon, USA) with the 100 µL medium containing 20% Matrigel (Corning, USA), 40 µM TS (or DMSO), and 0.1% FBS for each well. Under the cell permeable membrane, 600 µL of the complete medium was added for each chamber. After 24 h incubation, cells which have migrated through the membrane were fixed by 4% paraformaldehyde and dyed with crystal violet. The surface of upper layer of membrane was cleaned softly by neat cotton swabs, and the cells in different fields of view on the subface were counted by using ImageJ (version 1.52a; National Institutes of Health, USA) with triplicate.
For evaluating the effect of
TS on proliferation of TNBC cells, 3D spheroid formation assay was performed as described elsewhere [
17]. The working concentration of
TS was 40 µM. One set was used for capturing images from 24 to 96 h incubation time, and another set was for in vitro bioluminescence signal determination by transferring to a 96-well plate in the presence of D-luciferin (150 µL/mL) (PerkinElmer, USA). The proliferation inhibition rate was calculated following the formula as: inhibition rate = (1-Absorbance of treated sample /Absorbance of control sample )×100%.
Mouse models and in vivo fluorescence imaging
Six-week female athymic nude mice (18–21 g) (n = 8) were purchased from Gempharmatech (Nanjing, China). Experimental protocols were approved by the Beijing University of TCM Institutional Animal care and Use Committee (No.104,195,489,042) and adhered to the NIH Guide for the Care and Use of Laboratory Animals. A freshly prepared mixture (50 µL complete culture medium, 5 × 10
5 MDA-MD-231
Luc, 50 µL Corning Matrigel Matrix HC, Phenol Red-free, and LDEV-free) was directly injected into lower right mammary fat pad (MFP) of each mouse under inhalation isoflurane anesthesia [
15,
18,
19]. All mice were then maintained for 7 days and examined by In Vivo Imaging System (IVIS) Lumina LT Imaging System (Xenogen/Caliper Life Sciences, USA) with Living Image 4.3.1 software (Caliper Life Sciences, USA). All mice were then divided into
TS group (n = 4) and NC group (the same final concentration of DMSO) (n = 4).
TS was started to orally administer at 45 mg/kg body weight to mice once every 2 days. Images of tumor sizes were collected every week. Mice were monitored for 50 days to collect survivorship data. Mice were sacrificed in a CO
2 chamber when ethically necessary due to clinical symptoms or substantial loss in body weight. To compare survival differences between
TS treated and control groups, log rank (Mantel-Cox) test was used for survival analysis in GraphPad Prism (version 9.1.1).
Statistical analysis
Each assay was performed at least 3 independent experiments with triplicate. Means and standard deviations were calculated, and the differences between groups were analyzed using a generalized linear model with post-hoc Tukey test for the correction of multiple comparisons. The two-tailed unpaired student’s t-tests were used for the difference comparison of two groups. Statistical significance was considered when P < 0.05 (two-sided). Kaplan-Meier Survival curve with a log-rank test was applied for survival curves.
Discussion
The addition of new “bullets” in the treatment of breast cancer may accelerate the cure of the disease, given that the combination of different regimens or therapies substantially prolongs breast cancer survivors.
TS (TS), a compound from several plants such as Tribulus terrestris and Agrimonia pilosa ledeb [
20], belongs to a saponin of the herb. Herbal saponin was reported to possess therapeutic effects against cardiovascular diseases, nervous system disorders, asthma, arthritis and diabetes [
21]. Our previous study has reported the anti-cancer effects of
TS on hepatocellular carcinoma as a CAXII inhibitor [
9]. In this preclinical study, we extended our previous study and showed its inhibitive effects against TNBC.
Carbonic anhydrases (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate and protons. Overexpression of CAXII (CA12) has been found in hypoxia—the oxygen deprivation of cancer cells due to a combination of poor tumor vascularization and high proliferation rate, despite the fact that hypoxia response elements (HREs) essentially lack in the 5’-upstream genomic region of CAXII gene. Moreover, overexpression of transmembrane CAXII in cancer has been found to be associated with cancer progression including rapid tumor growth and invasion, infiltration of surrounding normal tissues and the formation of metastases [
22]. Moreover, selectively silencing of CAXII gene in TNBC cell line MDA-MB-231 could lead to the supression of cell migration and invasion by interefering the p38 mitogen-activated protein kinases (p38 MAPK) signalling pathway [
10]. Similarly, the reduction of CAXII expression by blocking Hedgehog signaling pathway in MDA-MB-231 cells could limit the migration ability of cancer cells, which highlighted the potential role of CAXII to be an effective therapeutic target in TNBC treatment.
In line with the previous studies, in this study, we found that expression of
CAXII in clinical breast-cancer samples was significantly higher than in breast-normal tissues (Fig.
1A and B). Such phenomenon is also reproducible in our RT-qPCR assay that TNBC cells (MDA-MB-231 and BT-549) possess significantly higher level of
CAXII expression than in breast normal cell (MCF-10 A) (Fig.
2A). In addition, a worse overall survival was also found in patients with high expression of
CAXII. Furthermore, we also applied U-104 (SLC-0111), a potent specific carbonic anhydrase (CA) inhibitor for CAXII, to evaluate the inhibitive specificity of
TS as a positive control, as we did in our previous study[
9]. In both MDA-MB-231 and BT-549 cells, CAXII esterase activity assay showed that
TS could suppress CAXII esterase activity in a dose-dependent manner similar to the U-104 groups, which further revealed the inhibitive effect of
TS on CAXII. These observations suggest that CAXII is a potential therapeutic target, and may be exploited for the treatment of TNBC.
Cellular acidosis was reported to be one of triggers of apoptosis, therefore a high level of pHi was thought to prevent cancer cells from apoptosis. Evidence has also displayed that pHi reduction can cause cellular apoptosis by activating endonuclease II and MMP impairment [
23]. Furtheremore, caspase activation and mitochondrial depolarisation have been found to be activated by cytochromic acidification, staurosporine and ultraviolet light. Therefore the agents, including CAXII inhibitor, which can reduce the pHi in cancer cells, were thought to be pro-apoptotic by impairing intracellular homeostasis and metabolism of cancer cell [
24]. Additionally, the accumulation of the metabolic byproduct lactate and extracellular acidification have been reported to accelerate tumor cell proliferation, metastasis, and angiogenesis. Lactate acidosis has been found to reduce immune evasion in TNBCs [
25,
26]. Therefore, extracellular lactate reduction has been considered as an anti-cancer approach to enhancing the therapeutic effect of immune therapy[
27]. CAXII has been reported to maintain pH and CO
2 homeostasis, thereby affecting cancer progression, invasion, and resistance to therapy [
8]. In this study, it is the first time to display that
TS could not only significanly reduce the gene expression and activity of CAXII, but also increased pHe level and suppressed extracellular lactate and pHi levels, which creates a hostile microenviroment to block TNBC progressions. It also indicates that
TS possesses regulatory capacity for cellular acidosis which specifically possessed by CAXII inhibitors [
8].
TS showed significantly strong inhibitive effects on the proliferation, invasion and 3D spheroid formation of two TNBC cell lines, while had a limited impact on the normal cells. As for in vivo experiments, our results show that mice receiving TS treatment experienced a significantly decreased tumor burden (> 40% after 14d), and also had a significantly improved survival rate, indicating the efficacy of TS in TNBC treatment. The results of 3D formation assay, and intracellular or extracellular PH values testing assays indicate that TS could shape an effective antitumor microenvironment.
E2F1 and
E2F3 are oncogenes and have negative correlations with breast cancer patient survival [
17].
E2F1 has been reported to affect cell growth by regulating NF-κB, thereby enhancing tumor proliferation and anti-apoptosis. It can also cause immune escape by blocking the transcription of ICAM-1 [
28]. Meanwhile,
E2F3 expression appears to provide a growth advantage to tumor cells by activating cell proliferation [
29]. Overexpression of
E2Fs is frequently observed in advanced cancers and aggravates chemoresistance. Overexpress of
E2F1 and
E2F3 were found in breast adenocarcinoma when compared to normal breast tissues [
17]. In addition, Caspase3 (CASP3), as an apoptosis executive factor, not only can regulate many physiological processes including cancer stemness and autophagy, but also affect pyroptosis by mediating GSDM [
30]. Lack of CASP3 can lead to the resistance of cells to microenvironmental stress and treatments, thereby promoting tumorigenesis [
31]. Our previous studies have shown that
E2F1/E2F3/Caspase-3 axis, which is associated with multiple cellular physiological processes, such as cancer stemness and apoptosis, was an effective anti-cancer pathway in liver cancer and breast cancer (including TNBC), and that
TS had a positive effect on regulating such axis in HCC [
9,
15,
17]. Again, in this study, we demonstrated that
TS significantly inhibited the expression of
E2F1 and
E2F3, and increased CASP3 activity in both TNBC cell lines cultured in 2D and 3D systems.
Interestingly, we also found that
TS as CAXII inhibitor also could reduce the expression of CAXII, E2F1/E2F3 expression. This finding suggests that a positive feedback loop may exist. It has been shown that low pH levels under hypoxia conditions could induce CA expression [
7], and that acidic microenvironments could stimulate E2F pathway to promote tumor metastasis [
32,
33].
Limitations exist in this study. We uncovered the anti-cancer effects of
TS on TNBC in vitro and in vivo with a focus on targeting CAXII only. Since isoforms exist for CA, and one of which the isoform CAII ranks the third predicted targets of
TS, we cannot rule out the possibility of inhibitive effect of
TS on CAII, as a cancer malignant behavior regulator [
9,
34,
35]. Therefore, it will be interesting to further investigate the expression profile of other CA isoforms and relevant molecular docking in future studies for further digging the potential mechanisms of anticancer effects exerted by
TS. Moreover, in addition to further screening for more optimal working concentration, nanoparticles should also be applied to further avoid the risk of potential cytotoxicity of
TS in future study. Our previous study has shown that nanoparticle LNP-DP1 efficiently alleviated the cytotoxicity of
TS for normal cells [
15]. To further confirming and evaluating the anti-metastasis potential of
TS on TNBC cells, multiple positions of IVIS scanning combined with histological or genetic analysis of metastatic lesions shall be investigated in future study [
36].
In summary, we demonstrate that TS suppresses TNBC development by acting as a novel CAXII inhibitor in this study. To our knowledge, this is the first study to show the inhibitive effect of TS on TNBC, low-toxic property of TS on normal breast cell and the regulation of TS on E2F1/E2F3/Caspase-3 axis. Our results indicate a novel chemotherapeutic TS with CAXII functionality as a potential choice for TNBC treatment. Further investigation of TS is warranted to explore its therapeutic potential for the treatment of TNBC and other potential anti-cancer pathways.
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