Abstract
Tumor metabolism is a crucial aspect of cancer development, and mitochondria plays a significant role in the aggressiveness and metastasis of tumors. As a result, mitochondria have become a promising therapeutic target in cancer treatment, leading to the development of compounds known as mitocans. In our group, we have consolidated the search of anticancer therapies based on natural products derived from plants, obtaining extracts such as P2Et from Caesalpinia spinosa and Anamu-SC from Petiveria alliacea, which have been shown to have antitumor activities in different cancer models. These extracts, due to their complex molecular composition, can interfere with multiple functions during tumor progression. To better understand how these natural products operate (P2Et and Anamu-SC), we constructed a model using 4T1 murine breast cancer cells with reduced expression of genes associated with glycolysis (Hexokinase-2) and mitochondrial function (Cqbp). The results indicate that the cells were more sensitive to the Anamu-SC extract, showing significant decreases in glucose consumption, ATP production, and oxygen consumption rate. Additionally, we observed changes in mitochondrial function, which reduced the cells’ ability to migrate, particularly when C1qbp was silenced. This triple-negative breast cancer model allows us to identify potential natural products that can modulate tumor cell metabolism.
Introduction
The metabolism of tumor cells as a hallmark of cancer was described 100 years ago by Otto Warburg1, identified an increase in glucose fermentation even in the presence of oxygen, which suggested mitochondrial dysfunction. It is now known that mitochondrial metabolism is essential for the survival and migratory potential of different tumor clones within the tumor2. Hanahan and Weinberg listed a series of “cancer hallmarks”, among which energy imbalance is one critical point in tumor progression3,4. Indeed, tumor cells adapt their energy metabolism to changing conditions, ensuring efficient nutrient use and allowing different metabolic sub-phenotypes to coexist within the same tumor, ultimately promoting growth5.
Aerobic glycolysis is the predominant metabolism in cells with a high proliferative rate, facilitating the biosynthesis of macromolecules through intermediates like those from the pentose phosphate pathway, which generates nucleotides for DNA synthesis and NADPH for regulating oxidative stress6. On the other hand, mitochondrial metabolism through tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS), integrating anabolic and catabolic pathways, provides energy support from different energy sources such as glutamine and lipid beta-oxidation, by this means tumor cells can generate amino acids, fatty acids, and nucleotides, allowing the production of metabolites as intermediary substrates. In this sense, OXPHOS supports progression and metastasis in different cancer models5,7,8.
Hexokinase, a key enzyme in glycolysis, phosphorylates glucose to form glucose 6-phosphate (G6P), marking an essential and irreversible step in metabolism8. The expression of hexokinase isoform 2 (Hk-2) expression rises during tumor formation, enhancing glucose absorption and providing the cell with energy and access to many intermediary substrates for proliferation. Hence, inhibition of glucose uptake by pharmacological mechanisms such as 2-deoxyglucose (2-DG) or RNA interference induced a reversal of tumorigenesis, leading to a compensatory increment of OXPHOS metabolism9,10. Additionally, the C1qbp (P32) protein, which regulates mitochondrial function11 participates in the process of mitochondrial biogenesis and is reported as overregulated indifferent types of cancer; supports OXPHOS by interacting with the pyruvate dehydrogenase (PDH) enzyme. Inhibiting C1qbp leads to metabolic shifts that increase glycolysis while decreasing OXPHOS in various cell models11,12.
Triple-negative breast cancer (TNBC) exhibits a heterogeneous metabolic phenotype with an exacerbated glycolytic metabolism due in part to an increased expression of glycolysis-associated receptors and enzymes such as GLUT1, HK-2, and LDH, among others. There are numerous metabolic pathway-based subtypes with varying sensitivities to drugs that function as metabolic inhibitors; each also has a unique clinical outcome, with the high glycolytic subtype tumor having the poorest prognosis13,14,15,16. The treatment of TNBC depends on the molecular subtype, and the most common therapies include surgery, radiation therapy, and chemotherapy, with taxanes and anthracyclines being the most used17. Nevertheless, relapse and poor prognosis remain the Achilles heel in this type of tumor and have become a challenge in drug discovery18.
The search for new drugs that target the metabolism has increased in recent years, and among these, various plant extracts have been shown to influence different metabolic pathways19,20,21. Natural compounds potentially operate as enzyme inhibitors, directly targeting the enzyme’s function or impacting genes and oncogenes involved in the glycolytic system22. Flavonoids like curcumin, resveratrol, quercetin, and EGCG can reduce the expression of glycolytic enzymes in various tumor models19,20,21,23. On the other hand, natural products can also target mitochondrial functions, including the TCA cycle, ETC, and OXPHOS, making them potential “mitocans”, for cancer treatment. This mitocans are described as a collection of compounds that demonstrate anti-cancer effects through their interactions with molecular targets in mitochondria24,25,26,27,28. Terpenoids from various plants, such as Bombax ceiba, Gardenia jasminoides, Alisma orientale, or fruits, such as grapes and red fruits, are some of these natural products that can alter mitochondrial function and have an anti-tumor effect. Other natural products that can do the same include types of flavonoids and saponins24,29. In TNBC, the OXPHOS pathway is overactivated, and its dysfunction impacts cancer cell motility and increases chemotherapy sensitivity29,30,31.
We have previously obtained and described a flavonoid-rich extract called P2Et obtained from the plant C. spinosa, which showed cytotoxic activity accompanied by an alteration in mitochondrial membrane potential, an increase in cytoplasmic calcium levels, damage-associated molecular pattern molecules (DAMPs) exposure in the cell surface, and induction of immunogenic cell death32,33. Similarly, from P. alliacea, we obtained the extract Anamu-SC, rich in flavonoids and sulfur compounds34. Treatment in 4T1-tumor cells showed a metabolic modulating effect with decreased glycolytic activity and decreased expression of the mitochondrial complex V in a triple-negative breast cancer35,36. In this context, we wanted to delve into the impact of each of these extracts and enhance our understanding of the biological function of polymolecular drugs, particularly regarding the participation of cell metabolism in controlling tumor cell death or migration. To accomplish this, we generated clones of the mouse breast cancer cell line 4T1 with a diminished expression of either the hexokinase-2 enzyme or C1qbp, showing alterations in glycolysis or OXPHOS, respectively. We assessed the relationship between these metabolic phenotypes in response to therapy with these natural extracts.
Methods
Plant material and natural extracts production
Leaves and stems from Petiveria alliacea L., Phytolaccaceae (local name “anamu”) were collected in Cachipay, Cundinamarca, Colombia and identified by Antonio Luis Mejia from the Colombian National Herbarium; voucher number COL 569,765 (Colombian Environmental Ministry agreement number 1927 related to these of genetic resources and derivatives products). P. alliacea extraction procedure and chemical characterization were previously described37,38.
Caesalpinia spinosa (Molina) Kuntze pods were collected in Villa de Leyva, Boyacá, Colombia. Plant material was identified by Luis Carlos Jimenez, from the Colombian National Herbarium (voucher specimen number COL 523,714. Colombian Environmental Ministry agreement number 220/2018 related to the use of genetic resources and derived products). The P2Et extract was produced under GMP conditions and chemically characterized as previously described39,40. In each assay, P2Et was diluted in 95% ethanol, obtaining a 25 mg/mL fresh solution.
Cell line and culture conditions
The 4T1 wild-type (WT) murine mammary carcinoma cell line (ATCC-CRL-2529) was originally provided by Dr. Alexzander Asea from Texas A&M Health Science Center College of Medicine in Temple, TX37,41. In the laboratory, the cell line was initially activated by inoculation into Balb/c mice, then recovered and cultured, and used between passage 3 and 10 for the experiments in the article. This cell line served as the parental line for the derivative clones 4T1-F10 (sh-Hk2)9 and 4T1-C12 (sh-C1qbp)11. The cells were cultured in RPMI-1640 (Gibco, Waltham, MA, USA) enriched with; penicillin (100 U/mL), HEPES buffer (0.01 M), l-glutamine (2 mM), sodium pyruvate (1 mM) and streptomycin (100 μg/mL) (Gibco, Waltham, MA, USA) including 10% heat-inactivated fetal bovine serum (FBS) (Eurobio, Les Ulis, France). The cultures were maintained in a humidified atmosphere at 37 °C with 5% CO2. Cells were allowed to grow to approximately 80% confluence before passaging using trypsin–EDTA. Additionally, the 4T1-F10 and 4T1-C12 cells were maintained with 10 μg/mL of puromycin (Gibco, Waltham, MA, USA).
HK-2 and C1qbp shRNA knockdown
The shRNA lentiviral particles targeting Hexokinase 2 (sc-35622-V), and C1qbp (sc-42881-V) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA USA). 2.5 × 104 4T1 cells seeded on 48 well plate were transduced with 106 virus particles in RPMI-1640 (Eurobio) supplemented with 10% FBS (Eurobio), 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, and 1 mM sodium pyruvate (Eurobio, Les Ulis Cedex B, France). Then, 5 μg/mL of polybrene was added and transduction was done in a humidified environment at 37 °C and 5% CO2 following the manufacturer’s instructions. Fresh medium was added every other day and cells were cultured in the presence of 10 μg/mL puromycin to select resistant cells (Gibco, Waltham, MA, USA). After 90% cell confluence, cells were trypsinized, expanded, and a monoclonal cell population was obtained throughout limiting dilution. After expanding different clones, the efficacy of the lentivirus-mediated shRNA knockdown of Hk-2 and C1qbp was confirmed by Western blot.
HK-2 and C1qbp expression analysis by western blotting
Cells were trypsinized, washed twice with PBS and lysed on ice for 20 min with RIPA buffer (150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate). Lysates were centrifuged, and protein concentration was measured using the Pierce BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Scientific, Massachusetts, USA). A total of 20 μg of protein were separated on a 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in (Tris-buffered saline with 0.1% Tween® 20 detergent) TBST-T supplemented with 5% nonfat dry milk for 1 h at room temperature. The primary antibodies rabbit mAb anti-hexokinase II (C64G5, Cell Signaling, Massachusetts, USA), rabbit mAB anti-C1qbp (D7H12, Cell Signaling, Massachusetts, USA) and monoclonal mouse IgG1 anti-β-actin antibody (MAB8929 from R&D systems, Minneapolis, USA) were incubated overnight at 4 °C, followed by specific secondary antibody, goat anti-rabbit IgG (H + L) secondary antibody HRP-conjugated (Novus, USA) and goat anti-mouse IgG antibody (H + L) HRP conjugate (Merck, Darmstadt, Germany). Membrane-bound immune complexes were visualized using SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) in an iBright FL1500 imaging system (Thermo-Fisher, Massachusetts, USA).
OCR and ECAR evaluation
The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Agilent MitoXpress and pH-Xtra assays, respectively, following the manufacturer’s protocols46,47,48 as was described before49. In this study, 4T1-WT, 4T1-F10, and 4T1-C12 cells were plated in a 96-well format at a density of 3.0 × 104 cells per well and treated for 6 h with the IC50 concentrations of P2Et or Anamu-SC as indicated by the MTT assay results (Fig. 5A). The controls of this type of experiments were added before the measure in the Cytation5 Reader (BioTek, Winooski, VT, USA). In this order, the OCR control was 1 μM AntiA (Antimycin A from Streptomyces sp, Sigma-Aldrich, Massachusetts, USA); and for the ECAR was used 50 mM 2-DG (2-deoxy-d-glucose, > 98%, Sigma-Aldrich, Massachusetts, USA). Afterwards, the measure of fluorescence decay rate was made it as the manufacturer’s protocol using the formula: Lifetime (µs)[τ] = (D2 − D1)/ln(IW1/IW2). The values of IW1 and IW2 are the two measurement windows; and D1 and D2 are the delay times before having the values of W1 and W2.
Glucose uptake assay
The glucose uptake assay was conducted using the fluorescent glucose analog 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (Invitrogen™/Molecular Probes, Carlsbad, CA, USA) and analyzed by flow cytometry50,51 and described previously42. The procedure was as follows; 1 × 105 4T1-WT, 4T1-F10, and 4T1-C12 cells were plated in 12-well plates and incubated overnight. Then, the cells were treated for 24 h with the IC50 concentration of P2Et, the IC50 and IC50/5 concentrations of Anamu-SC, 100 nM ascorbic acid (as an antioxidant control), 1 µM rotenone (a mitochondrial inhibitor), and either H2O or ethanol (as negative controls at 0.02%). To continue the cells were first stained with LIVE/DEAD fixable aqua kit to identify viable cells; subsequently, cells were incubated with 25 µM 2-NBDG in PBS for 30 min at 37 °C. After washing and resuspension in PBS, samples were analyzed using a Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA). FlowJo software v10.8.1 software (BD Life Sciences, New Jersey, USA) was used to analyze the flow cytometry data. Experiments were performed in duplicate on three independent experiments and the results were expressed as mean ± SEM.
ROS measurement
To evaluate ROS production, a total of 1 × 105 4T1WT, 4T1-F10 and 4T1-C12 cells were seeded on 12-well plates and incubated overnight. Then cells were treated with the IC50 of the P2Et, the IC50 and IC50/5 of Anamu-SC; 100 nM Ascorbic acid, 1 µM rotenone and H2O or ethanol (negative controls, 0.02%) for 24 hours. Then cells were labelled with 1 µM 2′,7′ dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma Aldrich, Massachusetts, USA) for 40 min at 37 °C followed by propidium iodide (PI) (Sigma-Aldrich, Massachusetts, USA) or 1 µM MitoSOX™ Red Mitochondrial Superoxide (Molecular Probes, Invitrogen Corp, Carlsbad, CA, USA) for 30 min at 37 °C. Each sample was then acquired using an FACSAria II-U flow cytometer (BD, Biosciences, San Jose, USA) for H2DCFDA and Cytek Aurora flow cytometer (Cytek) for MitoSOX. Data were analyzed with FlowJo v10.8.1 software (BD Life Sciences). Experiments were performed in duplicate on three independent experiments and the results were expressed as mean ± SEM.
ATP determination
The intracellular ATP analysis was performed using the ATP Bioluminescence Assay Kit HS II from Roche, following the manufacturer’s protocol explained before33,49. The experimental procedure was as follows: 1 × 105 cells of 4T1-WT, 4T1-F10, and 4T1-C12 were plated in 12-well plates and incubated overnight. The cells were then treated for 24 h with the IC50 concentration of P2Et, the IC50 and IC50/5 concentrations of Anamu-SC, 100 nM ascorbic acid, 1 µM rotenone and either H2O or ethanol (as negative controls at 0.02%). Following, the signal was capture in the Cytation5 Reader (BioTek, Winooski, VT, USA). The experiments were conducted in duplicate across three independent trials, and the results were reported as mean ± SEM.
Growth rate assay
4T1-WT, 4T1-F10, and 4T1-C12 cells were plated in 12-well plates at a density of 4000 cells per cm2. After 24, 48, and 72 h, the cells were harvested and counted using 0.4% trypan blue. The population doubling time (PDT) was determined using the exponential growth method (Malthusian) through GraphPad Prism version 8.1.1 for Mac OS X statistics software (GraphPad Software, San Diego, CA) described before49.
In vitro cytotoxicity assay
The methylthiazol tetrazolium (MTT) assay (Sigma-Aldrich, Massachusetts, USA) was utilized to assess the viability of tumor cells treated with plant extracts and doxorubicin detailed previously37,40. For each experiment, 4 × 103 cells were plated in 96-well plates. Treatments were given for 48 h in serial dilutions, starting from 250 μg/mL and decreasing to 0.97 μg/mL. After the treatment period, 100 μL of non-phenol red media and 50 μL of 1X MTT were added to each well, followed by a 4-h incubation at 37 °C. Subsequently, 100 μL of DMSO was added to dissolve the formazan crystals, and the plates were incubated for an additional 20 min. The absorbance was then measured at 540 nm using a Multiskan™ FC Microplate Photometer (Thermo Scientific, Massachusetts, USA). The IC50 values (50% inhibition of cell growth) were calculated using GraphPad Prism version 8.1.1 for Mac OS X, employing a non-linear regression log (inhibitor) versus response-variable slope formula. Experiments were conducted in triplicate across three independent trials, with results expressed as mean ± SEM.
Annexin V and PI double-staining assay
Phosphatidylserine (PS) externalization was assessed using flow cytometry with Annexin V-FITC (Molecular Probes, Invitrogen Corp, Carlsbad, CA, USA) and propidium iodide (PI) (Sigma-Aldrich, Massachusetts, USA) staining54,55 and analyzed by flow cytometry following the previously mentioned protocol40. The protocol involved seeding 1 × 105 cells (4T1-WT, 4T1-F10, and 4T1-C12) in 12-well plates and incubating them overnight. Cells were then treated for 24 h with various compounds, including IC50 of P2Et, IC50 and IC50/5 of Anamu-SC, 100 nM ascorbic acid, 1 μM rotenone, and controls (H2O or 0.02% ethanol). After treatment, cells were harvested and resuspended in annexin buffer (100 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2)55. The cell suspension was incubated with Annexin V-FITC for 8 min at room temperature, followed by a 3 min incubation with PI at 4 °C. Data acquisition was performed using a FACSAria II-U flow cytometer, and the results were analyzed using FlowJo v10.8.1 software.
Mitochondria membrane potential (MMP) assay
To evaluate MMP, 1 × 105 4T1-WT, 4T1-F10 and 4T1-C12 cells were seeded on 12-well plates and incubated overnight. Then cells were treated for 24 h with the IC50 of the P2Et, the IC50 and IC50/5 of Anamu-SC; 100 nM Ascorbic acid (anti-oxidant), 1 µM rotenone (mitocan), valinomycin (positive control, 0.1 μg/mL) and H2O, or ethanol (negative controls, 0.02%) for 24 h. MMP was measured using JC-1 (Sigma-Aldrich, Massachusetts, USA) dye. The cells were acquired on a FACSAria I (Becton, Dickinson and Company, New Jersey, USA) and analyzed with FlowJo software (Tree Star Inc., Ashland, OR), which calculated the red/green fluorescence ratios. Duplicate estimations were made, and the average was expressed as mean ± SEM in three independent experiments.
Mitochondrial density and morphological analysis
For mitochondrial density, 1 × 105 4T1-WT, 4T1-F10 and 4T1-C12 cells were seeded on 12-well plates and incubated overnight. Then cells were stained with 0.2 µM of MitoTracker Red CMXRos (Molecular Probes, Invitrogen Corp, Carlsbad, CA, USA) for 30 min at 37 °C. Each sample then was acquired using a Cytek Aurora flow cytometer (Cytek Biosciences). The samples were analyzed with FlowJo v10.8.1 software (BD Life Sciences). Experiments were performed in triplicate and the results were expressed as mean ± SEM.
Mitochondria morphological parameters were evaluated by immunofluorescent microscopy. 2 × 104 4T1-WT, 4T1-F10, and 4T1-C12 cells were seeded on a 35 mm glass bottom dish with 10 mm micro-well treated with 0.1% fibronectin and were grown overnight in 1 mL of RPMI-1640 medium. Subsequently, they were treated for 24 h with IC50 of Anamu-SC: 125 μg/mL for 4T1-WT, 121.6 μg/mL for 4T1-F10 and 60.39 μg/mL for 4T1-C12 and 1 μM rotenone for 5 min. At the end of time, cells were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde for 20 min, then washed and permeabilized with 0.1% Triton X-100 for 10 min. Next, blocking was carried out with 50 mM NH4Cl for 10 min, followed by incubation with MitoTracker™ Red CMXRos (Molecular Probes, Invitrogen Corp, Carlsbad, CA, USA) for 20 min at 37 °C. Finally, cells were stained with 300 nM DAPI (Molecular Probes, Invitrogen Corp, Carlsbad, CA, USA) for 5 min. Images were acquired with an Olympus FV1000 confocal microscope, with a 60 × PlanAPO oil objective. Quantitative mitochondrial analysis was performed on 50 cells per coverslip according to the protocol described by Koopman et al.56 and Tronstad et al.57 using ImageJ software 1.52 q. Parameters calculated from the images for each condition were: the number of mitochondrial objects per cell (Nc), the mean area of each individual mitochondrial object (Am), mitochondrial mass (the product of Nc and Am), the mean mitochondrial object aspect ratio (AR, a measure of mitochondrial length), and the mean mitochondrial object shape/form factor (FF, a combined measure of mitochondrial length and degree of branching).
Wound healing assay
To assess cell migration, a scratch wound assay was performed using a standard pipette tip to create gaps in the cell monolayer58,59. The experiment also described before43 utilized 4T1-WT, 4T1-F10, and 4T1-C12 cells, which were initially seeded at a density of 1 × 105 cells per well in 12-well plates and allowed to adhere overnight. The cells were then exposed to IC50/5 concentrations of P2Et and Anamu-SC treatments. Following a 48-h incubation period in RPMI-1640 supplemented medium, the cells were re-seeded in 12-well plates and cultured until they formed a confluent monolayer (approximately 90% confluence). Once confluence was achieved, the cell monolayer was wounded by scratching with a pipette tip, creating a cell-free area. The wounded monolayers were then cultured in fresh Opti-MEM I Reduced Serum Media. To monitor wound closure, images were captured at the initial time point and at 12- and 24-h post-wounding43. The cell migration was documented using a low magnification (4 ×) objective on a BioTek Cytation 5 Cell Imaging Multimode Reader (BioTek, Winooski, VT, USA). Quantitative analysis of wound size measurements was performed using ImageJ software, a widely used tool for such analyses.
qPCR of EMT transcription factors
The TRIzol LS reagent (Life Technologies Corporation, Invitrogen, NY) was used to have the total RNA from 4T1-WT, 4T1-F10, and 4T1-C12 cells according to the manufacturer’s protocol and previously described43. The process began by seeding 1 × 105 cells overnight, followed by resuspension in TRIzol. cDNA synthesis was performed using SuperScript III Reverse Transcriptase (Invitrogen) as per the manufacturer’s guidelines. RNA quality and quantity were evaluated using a NanoDrop spectrophotometer (NanoDrop Technologies). For real-time PCR, reactions were set up in a total volume of 20 µL, containing 600 ng of cDNA, iTaq Universal SYBR Green Supermix (BIORAD, CA, USA), and 250 nM of both forward and reverse primers. To assess the expression of Snail, Twist, and Zeb genes, the following specific primers were utilized (Table 1):
Data was collected from two independent experiments, each performed in duplicate, using the QuantStudio™ 3 Real-Time PCR System (Molecular Probes, Invitrogen Corp, Carlsbad, CA, USA). The PCR thermal cycling program consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 15 s; annealing at the appropriate temperature for 60 s and extension at 72 °C for 40 s, finally, the program concluded with a dissociation phase to verify amplification specificity.
Gene expression levels were normalized to the housekeeping gene GAPDH, which served as an endogenous control. Relative expression was then calculated using the 2^(− ΔΔCT) comparative method, allowing for quantitative comparison of gene expression across different samples and conditions.
Statistical analysis
For statistical analysis we employed between two groups was calculated using the Mann–Whitney U test, while differences among groups were calculated using Kruskal–Wallis and Dunn’s posttest for multiple comparisons. GraphPad Prism version 8.1.1 for Mac OS X statistics software (GraphPad Software) was used.
Results
Hk-2 downregulation in 4T1 cells reduces glucose uptake, ATP, and ROS production
We generated two stable clones with different metabolic characteristics through the knockdown of the Hk-2 and the C1qbp proteins in the murine breast cancer 4T1 cells transducing lentiviral particles containing three shRNA in a puromycin resistance vector. Thus, after transduction, we selected by limiting dilution and obtained the stable clones for each single protein: 4T1-F10 and 4T1-C12 clones for Hk-2 and C1qbp downregulation, respectively. Western blot analysis of the expression levels of Hk-2 in the F10 clone and C1qbp in the C12 clone confirmed that each protein’s expression was diminished by the shRNAs (Fig. 1A). Then, we examined glucose uptake, intracellular ATP production, mitochondrial mass, and intracellular/mitochondrial reactive oxygen species (ROS/mtROS). In the 4T1-F10 clone compared to the 4T1-WT, we observed a decreased glucose consumption, ATP generation, and mtROS with a tendency to increase mitochondrial mass (Fig. 1B–F). However, compared to 4T1-WT cells, the 4T1-C12 clone only showed a decrease in ATP generation; this could be because 4T1-WT cells have a high rate of glycolysis61.
Metabolic parameters evaluation in shRNA-Hk-2 and shRNNA-C1qbp 4T1 clones. (A) Western blotting showing protein Hk-2 and C1qbp expression of each clone, 4T1-F10 (Hk-2) and 4T1-C12 (C1qbp). (B) 4T1-WT, 4T1-F10 and 4T1-C12 clones were seeded and after 24 h were stained with 2-NBDG dye showing MFI (median fluorescence intensity). (C) 4T1-WT, 4T1-F10 and 4T1-C12 cell were seeded overnight then cells were lysed and supernatant recollected showing bioluminescence per 100.000 cells. (D–F) 4T1-WT, 4T1-F10 and 4T1-C12 cells were seeded overnight and then, stained with mitotracker CMXRos (D), H2DCFDA (E), and MitoSOX (F), figures showed MFI of each probe. Data are presented as the mean ± SEM of three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Hk-2 and C1qbp downregulation in 4T1 cells modulate glycolysis and OXPHOS metabolism
Then, we looked for variations in the ECAR and OCR between 4T1-WT cells and their clones. We found that only the 4T1-F10 clone significantly increased the OCR compared to 4T1-WT cells, whereas the ECAR significantly decreased in both clones (Fig. 2A). The response of 4T1-WT and its clones to the glycolysis inhibitor 2DG and the inhibitor of the mitochondrial electron transport chain, antimycin, was then assessed in ECAR and OCR, respectively. We observed that clone 4T1-F10, were sensitive to 2DG, lowering ECAR, and that both clones 4T1-1-F10 and 4T1-C12 lowered OCR in response to antimycin (Fig. 2B). The ECAR/OCR rate analysis showed that 4T1-WT and 4T1-C12 preferentially use glycolysis, while 4T1-F10 uses OXPHOS, suggesting that clone 4T1-F10 has a marked tendency to use this pathway, in fact, this clone showed a lower glucose consumption (Fig. 1B), as expected, whereas the 4T1-C12 clone was similar to 4T1-WT cells being more glycolytic (Fig. 2C)9,11. Besides, plotting OCR vs ECAR results we could separate the cells into different metabolic phenotypes (Fig. 2D) as previously shown in other models62. These findings demonstrate that, in this TNBC cell model, metabolic regulation via Hk-2 knockdown may rely to an OXPHOS-depending metabolic model, whereas C1qbp inhibition does not clearly distinguish itself from 4T1-WT cells.
ECAR and OCR differences in 4T1-WT, Hk-2 and C1qbp knockdown cells. (A–D) Cells were seeded in 96-well plate overnight, and next day cells were stained with MitoXpress probe, or pH-Xtra Glycolysis probe and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured. (A) ECAR and OCR basal measurement in 4T1-WT, 4T1-F10 and 4T1-C12. (B) Graph shows in Y left axis the ECAR fold change (dot red line) with the 2-DG 50 mM (glycolysis inhibitor) and Y right axis shows the OCR fold change (dot red line) with antimycin A 1 µM electron transport chain (ETC) inhibitor. (C) Graph shows the ratio between ECAR and OCR in the different cells. (D) Graph shows the metabolic profile of cells. Data are presented as the mean ± SEM of 3 independent experiments *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Hk-2 and C1qbp downregulation in 4T1 cells increase cellular migration
The alteration of different metabolic pathways at the tumor cell level is related to changes in proliferation index and cell migration63,64,65. To confirm this response, first, we wanted to compare the growth rate between the 4T1-WT cells and the clones through the measurement of the population doubling time (PDT). The 4T1-F10 clone proliferated at a slower rate than WT cells, while there were no alterations in the 4T1-C12 clone (Fig. 3A). Next, using the wound closure assay, we observed that both clones had a higher percentage of wound closure compared to WT cells, with no differences in the migration between the clones (Fig. 3B). Since tumor cell migration is related to changes in the epithelial-mesenchymal transition (EMT), a set of transcription factors associated with it were evaluated (Table 1). We found a significant increase in Zeb1 relative expression in the 4T1-C12 clone. Still, no variation was found between the WT cells and the 4T1-F10 clone (Fig. 3C). These results suggest that the ability to migrate is related to metabolic modulation, which may involve pathways other than the activation of EMT transcription factors.
Migration in 4T1 cells with deficit in Hk-2 and C1qbp. (A) Growth curve of 4T1-WT, 4T1-F10 and 4T1-C12 clones seeding at density of 4000 cells/cm2. Data are presented as mean ± SEM. (B) Cell migration images (wound healing assay) of 4T1-WT, 4T1-F10 and 4T1-C12 cells showed an increase in wound closure of the clones over WT cells with graph representation of percentage closure at 12 h and 24 h in 4T1WT cells and their clones. (C) Relative expression of the EMT transcription factors Zeb, Twist and Snail in 4T1WT cells and their clones, analyzed by the 2-ΔΔCt method compared with 4T1WT cells as control (red dash line). Data are shown as mean ± SEM *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Changes in mitochondrial function reduce the migration ability of 4T1-F10 and 4T1-C12
Mitochondria may be a therapeutic target for a group of drugs known as mitocans25,26, including substances such as rotenone that inhibit mitochondrial complex I66. Thus, we studied the effect of rotenone in the mitochondria membrane potential (MMP) on 4T1-WT cells and its derived clones. Rotenone treatment increased MMP loss in WT and clones, with the C1qbp knockdown clone being less sensitive, while the Hk-2 inhibition clone had the greatest loss, likely due to a reduction in glucose consumption (Fig. 4A). Then, after rotenone treatment, we measured ROS/mtROS from the cells and discovered an increase in both parameters in all cells, with mtROS being higher in WT cells (Fig. 4B). However, analysis of cell migration revealed a decrease in both clones, with clone C12 showing a higher decline. Since WT cells did not form a confluent cell monolayer in the wound healing assay after being treated with rotenone (Fig. 4C), the assay could not be carried out on them. Then, we wanted to see if the effect of rotenone on migration was connected to a metabolic aspect such as ATP production, and we discovered a significant reduction 24 h after treatment in 4T1WT cells and both clones (Fig. 4D). These findings suggest that mitocans therapy impairs mitochondrial function independent of metabolic fitness, and that this impairing affects tumor cell migration.
Mitocans decrease migration in both clones. (A) JC-1 staining analysis was done in cells seeded overnight and treated with 1 µM of Rotenone for 24 h. Percentage of JC-1 aggregates expressed as mean ± SEM of three independent experiments is shown. (B) 4T1-WT, 4T1-F10 and 4T1-C12 clones were treated with 1 µM of Rotenone and after 24 h were stained with H2DCFDA and MitoSOX showing MFI fold change compared with untreated cells (blue dot line). (C) 4T1-WT, 4T1-F10, and 4T1-C12 clones were treated with 0,1 µM rotenone for 48 h, percentage closure in 4T1-WT cells and clones is shown. (D) 4T1-WT, 4T1-F10, and 4T1-C12 clones were treated with 1 µM Rotenone, the graph shows the bioluminescence per 100.000 cells. Data are presented as the mean ± SEM of 3 independent experiments *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Anamu-SC reduced the 4T1-F10 and 4T1-C12 tumor cells migratory potential and altered tumor mitochondrial metabolism
To investigate whether natural extracts with antitumor activity exerted their function through the regulation of mitochondrial function, we evaluated the effect of P2Et and Anamu-SC on the mitochondrial-related biological functions: glucose consumption, ATP, and ROS production. First, we found that the 4T1-F10 clone presented with ten times more sensitivity to cell death when treated with Anamu-SC compared to 4T1-WT, and two times more than the 4T1-C12 clone. No differences were observed between WT cells and the clones in terms of their sensitivity to the P2Et extract (Fig. 5A). Therefore, in the following tests the IC50 of the extracts were calculated for each clone. We observed that Anamu-SC caused a decrease in glucose uptake in 4T1-WT cells and the 4T1-C12 clone. Still, it did not affect the 4T1-F10 clone, probably due to the inhibition in Hk-2 (Fig. 5B). In addition, and as it was expected, the Anamu-SC as well as the rotenone treatment caused a reduction in ATP production in the WT cells and clones35. On the other hand, the P2Et extract did not influence ATP production in the clones, but it did decrease ATP production in WT cells (Fig. 5C).
Natural extracts with effect on metabolism reduce migration. (A) IC50 of P2Et and Anamu-SC treatment for each cell line, data were normalized to the viability of controls (EtOH-treated, vehicle). (B) 4T1-WT, 4T1-F10 and 4T1-C12 clones were treated with the IC50 of the natural extracts and controls for 24 h, then they were stained with 2NBDG dye showing MFI (median fluorescence intensity). (C) 4T1-WT, 4T1-F10 and 4T1-C12 clones were treated with the IC50 of the plants extracts and controls for 24 h, the graph shows the bioluminescence per 100.000 cells. (D) 4T1-WT, 4T1-F10 and 4T1-C12 clones were treated with the IC50 of the plants extracts and controls for 24 h, then they were stained with H2DCFDA and MitoSOX Red (100 mM) showing MFI from each treatment relative to the vehicle for both dyes. (E) 30.000 cells were seeded of 4T1-WT cell line and their clones 4T1-F10 and 4T1-C12 in 96 well plate overnight, next day cells were treated with the IC50 of P2Et and Anamu-SC (SC) for 6 h and then the cells were stained with MitoXpress probe or pH-Xtra Glycolysis probe and OCR and ECAR were measured in Cytation 5. (F) 4T1-WT, 4T1-F10 and 4T1-C12 cells were treated with the IC50 of the plants extracts and controls for 24 h, a representative graph of percentage of apoptotic cells (sum of early and late apoptosis) is shown expressed as mean ± SEM for three independent experiments. (G) 4T1-WT, 4T1-F10 and 4T1-C12 clones were treated with 1/5 IC50 of P2Et and Anamu-SC (SC), Rotenone 0,1 µM and ascorbic acid (AA) 20 nM for 48 h and then performed cell migration assay (wound healing assay) and percentage closure in 4T1-WT cells and clones is shown. Data are presented as the mean ± SEM of 3 independent experiments *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
We performed a parallel analysis of ROS and mtROS production, expecting that Anamu-SC treatment would cause an increase in ROS due to mitochondrial impairment. Indeed, we found an increase in both parameters in all cells, with more mtROS production in the WT cells (Fig. 5D). Then, using the IC50 of P2Et and Anamu-SC, we evaluated the OCR and ECAR after 6 h of treatment. P2Et treatment did not produce any differences, while Anamu-SC generated a significant overall drop in OCR in 4T1-WT and their clones, with an increase in ECAR in the 4T1-C12 clone (Fig. 5E). Next, we examined how different treatments affected the induction of apoptosis and found that 4T1-C12 cells were more susceptible to apoptosis when exposed to Anamu-SC and rotenone than 4T1-WT and 4T1-F10 cells. P2Et did not trigger apoptosis in 4T1-F10 cells as effectively as it did in 4T1-WT cells (Fig. 5F and Fig. Suppl 1). We used the wound healing assay to determine how the extracts affected cell migration. We discovered that Anamu-SC treatment diminished migration in both clones, being significantly more robust in the clone with C1qbp inhibition and that outcomes are comparable with those with rotenone treatment (Fig. 5G). Instead, the P2Et therapy had no impact on the ability to migrate. These findings show that the C12 clone, which has a slightly lower mitochondrial mass and a reduced ECAR compared to 4T1-WT cells, it is more sensitive to mitocans like rotenone and Anamu-SC treatment, the latter of which also affects various metabolic features like glucose consumption, ATP production, as well as migration and apoptosis.
Anamu-SC treatment affects mitochondrial morphology
Considering that mitochondria is a molecular target of Anamu-SC35, and that our current results seem to confirm this fact, we assessed whether there were changes in mitochondrial morphology. To test this, we treated WT cells and clones (F10 and C12) with Anamu-SC for 24 h and analyzed different factors related to mitochondria morphology, such as number (Nc), area (Am), mass, length (AR) and mitochondrial branching (FF) (Fig. 6A–C). Significant alterations were generated by Anamu-SC and rotenone treatments in the parameters as Nc, Am, mass (Fig. Suppl 2), mitochondrial length (Fig. 6B), and mitochondrial branching (Fig. 6C). In fact, all the parameters were reduced with Anamu-SC treatment in all the cells, in fact, we observed a more significant reduction on the 4T1-WT and the 4T1-C12 cells compared to the 4T1-F10 cells, however the Nc parameter and the mitochondrial mass tended to decrease in 4T1-C12 cells.
Mitochondrial morphology is affected by Anamu-SC. (A) 4T1-WT, 4T1-F10 and 4T1-C12 clones were seeded overnight then were treated with Anamu-SC IC50 and rotenone 100 nM then were stained with Mitotracker deep red and DAPI (blue) for the nuclei. Images observed related to 4T1-C12 clone (merge, mitotracker deep red and Koopman filter). These images were acquired with confocal microscope Olympus FV1000 with an oil 60 × PlanAPO. Quantification of mitochondrial morphological changes, (B) length, and (C). mitochondrial branching in 4T1-WT, 4T1-F10, and 4T1-C12 treated with Anamu-SC and Rotenone for 24 h. Data are presented as the mean ± SEM of 2 independent experiments *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
On the other hand, Rotenone reduced the Nc in all cell lines, however, the Am parameter was affected only on the 4T1-WT and 4T1-C12 cells; and the mitochondrial mass and FF were diminished in the 4T1-WT cells and both clones as Anamu-SC did. Here, the Anamu-SC showed a major impact on the mitochondrial network, leading to a reduction in branching as well as in mitochondrial length. These results suggest that the mitochondria is a target of Anamu-SC and therefore this could be proposed as a standardized polymolecular extract acting as a mitocan.
Discussion
Tumor metabolism represents a complex collection of metabolic changes that arise in response to genetic and nongenetic determinants. Metabolic reprogramming meets the demands of cell proliferation; however, there is a certain degree of heterogeneity given by the metabolic dependencies of any given tumor cell5,67. Due to the recognition of the complexity and metabolic heterogeneity, we designed two cellular models of triple-negative breast cancer with different metabolic requirements. Subsequently, we evaluated their biological behavior and response to metabolic modulators targeting the mitochondria. On one hand, we inhibited Hk-2 expression, an enzyme that regulates glycolysis, and on the other, we altered the gene expression of C1qbp, which codifies for a multifunctional chaperone-type protein in the mitochondria. We were able to show that the inhibition of Hk-2, clone 4T1-F10, makes cells more dependent on mitochondrial activity. Tumor mitochondrial metabolism has a vital role in metastatic triple-negative breast cancer. Even if this type of tumor relies on glycolysis, the OXPHOS is increased in metastatic tumors as well in chemo-resistant. In fact, OXPHOS inhibition in relapsed tumors reduced progression and improved the efficacy of chemotherapy, which suggests that targeting mitochondrial functions such as energy production by OXPHOS may be a novel approach to enhance the effectiveness of various targeted therapies for TNBC31,68,69.
Indeed, due to the tumor’s dependence on energy for the many stages of the metastatic process, such as detachment, extravasation, invasion, and new niche development, metabolism, and tumor migration are receiving more attention. Metastatic cells suffer a reprogramming of their metabolism, which results in increased OXPHOX activity in distant metastases70. Metastasis can occur in different ways in the primary tumor, mainly as individual or collective processes. In individual processes, an increase in mitochondrial metabolism is seen, bringing the mitochondria to the forefront, where they provide more ATP molecules to support migration. In the collective process, a heterogeneous population is composed of a leader cell and follower cells, which have a different metabolism compared to individual migration because the leader cell depends on glycolytic metabolism, which allows it to have ATP more quickly than the followers due to the high efficiency of glycolysis to generate ATP molecules from one molecule of glucose71.
Besides, it has been demonstrated that natural compounds possess antimetastatic properties. In triple-negative breast cancer, apple polyphenol phloretin inhibits GLUT2 transporter, which suppresses the growth and metastasis of the malignancy72. Additionally, MC-4, an extract from Artemisia annua L., decreased the growth of cancer cells by suppressing the expression of GLUT1 and PKM2, key proteins on the glycolysis pathway. Furthermore, the combination with everolimus, an inhibitor of mTOR, reduced the metastatic process in advanced renal cell carcinoma73. Sulfur compounds are present in different natural extracts, as in the Anamu-SC extract presented here34; in fact, these organic sulfur compounds have shown epigenetic alterations that confer anticancer properties; also, different pathways where these sulfurs have an effect in cancer, such as a reduction in metastasis, a disruption in the cell cycle, and, inhibition in proliferation, among others, have been published74.
Although the mechanisms of migration and invasiveness are not yet well understood, the link between metabolism and migration has led to the active search for molecules that affect mitochondria (mitogens)26. Targeting processes such as ETC by inhibiting complex I induce cytotoxicity and increased sensitivity to chemotherapy, as was demonstrated in pancreatic cancer75. Equally important has been the mitochondrial dynamic study; in TNBC, mitochondria are smaller and fragmented than in non-triple negative breast cancers correlating with poor prognosis. Therefore, the focus of interest is to search for molecules that inhibit fission69; however, until now, there are no recognized molecules for this purpose.
The complex processes of mitochondrial activity greatly influence tumor cells’ behavior, survival, and growth. Our study demonstrates that Anamu-SC extract can modify metabolism by reducing cellular respiration and mitochondrial biogenesis and affecting mitochondrial morphology in breast cancer cells. These changes could explain its ability to decrease cell migration and its cytotoxic effect in cancer, suggesting that it acts as a "mitocan". On the contrary, P2Et extract does not result in appreciable changes to mitochondrial shape or metabolism. However, P2Et affected cellular respiration in a leukemia model but not mitochondrial morphology (unpublished data). Previous research on the B16 melanoma model showed that P2Et triggers the activation of the endoplasmic reticulum stress response, leading to the intracellular release of calcium, and potentially inducing the generation of markers associated with immunogenic cell death32; these results suggest that while both extracts exhibit anticancer effects they operate through distinct mechanisms, having Anamu-SC playing a significant capacity to interfere tumor metabolism and mitochondrial function.
Other authors have shown how natural products influence the migration of tumor cells through mechanisms distinct from those that are metabolic-related. Ferulic acid and coumarin derivatives can alter melatonin receptors, which generates an anti-migratory and antiproliferative effect on different breast cancer cell lines, and particularly curcumin can intervene in PI3K/Akt signaling, preventing cell migration of the MDA-MB-231 line76,77. In turn, Cordycepin, a nucleoside isolated from Cordyceps, negatively regulates mitochondrial function and limits energy production, leading to inhibition of metastasis and migration in ovarian cancer78,79. Also, lupeol, a pentacyclic triterpenoid compound, induces mitochondrial hyper-fission, eventually leading to apoptosis in renal carcinoma cells80.
In conclusion, we introduce a mouse model of triple-negative breast cancer (TNBC) where the tumor cell´s energy metabolism was modulated by the anti-tumor effect of two distinct plant extracts. The first, P2Et, known for its significant antitumor effect in melanoma, and leukemia models32,33,45,81,82,83, appears to have no impact on the metabolic pathways in this TNBC model. On the other hand, Anamu-SC, primarily focuses on targeting mitochondria, while also impacting various metabolic aspects, particularly the reduction in oxygen consumption rate (OCR), besides, it has been found with other extracts derived from P. alliacea the capacity to reduce respiration and disrupt vital metabolic pathways involved in the proliferation of leukemic cells49. This leads to an enhancement of the cytotoxic capabilities and a decrease in the migration ability, which could be principally beneficial in metastatic cells. Therefore, we propose the Anamu-SC plant extract as a promising mitocan.
Data availability
The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format, they are available from the corresponding author upon reasonable request.
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Acknowledgements
The authors would like to thank Pontificia Universidad Javeriana and the Colombian Environmental Ministry for allowing the use of genetic resources through the Contract of Access to Genetic Resources No. 220 of 2018 for Colombian plant material.
Funding
This project was funded by Pontificia Universidad Javeriana, Ministerio de Ciencia, Tecnología e Innovación, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo and ICETEX, 2ª Convocatoria Ecosistema Científico–Colombia Científica 792–2017, Program “Generación de alternativas terapéuticas en cáncer a partir de plantas a través de procesos de investigación y desarrollo traslacional, articulados en sistemas de valor sostenibles ambiental y económicamente” (Contract no. FP44842-221–2018). We thank the Vicerrectoria de Investigaciones at the Pontificia Universidad Javeriana for the financial support to publish this research (Proposal ID 00010639).
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Conceptualization C.C., S.F and A.B.; conducted experiments M.C.J., C.A., and C.C.; data collection and data analysis M.C.J., C.A., P.L., C.U and C.C.; data interpretation C.A., P.L., C.C., and A.B.; manuscript writing C.C., and A.B.; writing review and critical revision M.C.J., C.A., P.L., C.U., C.C., A.B., and S.F. Grant acquisitions S.F and AB. All authors have read and agreed to the published version of the manuscript.
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S.F. and C.U. are inventors of a granted patent related to P2Et. SF and CU are partners of the DreemBio company who was a licensee of related patents. The rest of the authors declare no competing interests.
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Carlosama, C., Arévalo, C., Jimenez, M.C. et al. Triple negative breast cancer migration is modified by mitochondrial metabolism alteration induced by natural extracts of C. spinosa and P. alliacea. Sci Rep 14, 20253 (2024). https://doi.org/10.1038/s41598-024-70550-z
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DOI: https://doi.org/10.1038/s41598-024-70550-z
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