Antiproliferative effects of 13α/β-steroids on triple-negative MDA-MB-231 breast cancer cells: unraveling intracellular signaling without ERα

Abstract This study aimed to investigate the activities of novel 20(R)-3,20-dihydroxy-19-norpregn-1,3,5(10)-trienes (kuz7 and kuz8b) of natural 13β- and epimeric 13α-series against triple-negative MDA-MB-231 breast cancer cells. High antiproliferative activity of synthesized compounds kuz8b and kuz7 against MDA-MB-231 triple-negative cancer cells was revealed. The steroid kuz7 of natural 13β-configuration was more active against MDA-MB-231 cells than the 13α-steroid kuz8b. Cell cycle analysis revealed common patterns for the action of both tested compounds. The number of cells in the subG1 phase increased in a dose-dependent manner, indicating induction of apoptosis, which was also verified by PARP cleavage. In contrast, the number of cells in the G0/G1 phase decreases with increasing compound concentration. Steroid kuz7 at micromolar concentrations reduced the expression of GLUT1, a glucose transporter. High efficacy of the combination of kuz7 with biguanide metformin was shown, and synergistic effects on MDA-MB-231 cell growth and expression of the anti-apoptotic protein Bcl-2 were revealed. According to the obtained results, including the high activity of kuz7 against triple-negative cancer cells, the detected induction of apoptosis, and the decrease in GLUT1 expression, 13β-steroid kuz7 is of interest for further preclinical studies both alone and in combination with the metabolic drug metformin.


INTRODUCTION
Cancer is a leading cause of death worldwide (de Martel et al., 2020).More than 19 million new cases of cancer were reported in 2020, and it has been estimated that this number will continue to rise.Breast cancer is the most common cancer among women and one of the leading causes of death among them.Breast cancer is a multifactorial disease (Momenimovahed, Salehiniya, 2019;Tao et al., 2015), and the incidence and progression of this type of cancer are due to a variety of factors including genetic, environmental, lifestyle and other factors (Feng et al., 2018).The development of new screening programs can detect breast cancer at an early stage, but despite this, there has not been sufficient success in the treatment of this disease.
Treatment approaches for triple-negative breast cancers are very limited.It is impossible to use hormone therapy or anti-HER2 therapy for TNBC patients (Yao et al., 2017).Triple-negative breast cancer is typically treated with a combination of surgery, radiation therapy, and chemotherapy.Chemotherapy is recommended for the vast majority of triple-negative and HER2positive breast cancers because these cancers are often very aggressive.The most commonly used treatment regimens involve anthracyclines and taxanes, although cyclophosphamide, methotrexate, and 5-fluorouracil may also be used in selected patients (Ntellas et al., 2019).Several studies have shown the efficacy of PARP inhibitors and immunotherapy for patients with triplenegative cancers (Barchiesi et al., 2021;Vikas et al., 2020).Although there are many drugs for triple-negative cancers, the effectiveness of treatment is not always high.Therefore, the development of new drugs and drug combinations for the treatment of triple-negative cancer is very relevant (Koushki et al., 2021;Roshanazadeh, Babaahmadi Rezaei, Rashidi, 2021).
Steroids are widely used in the treatment of hormone-dependent breast cancer.The estrogen receptor inhibitor fulvestrant and several aromatase inhibitors are highly effective in the treatment of ERα-positive cancer.In preclinical studies, steroids and their analogs have been shown to exhibit significant antiproliferative potency against triple-negative cancer cells.Several compounds are shown in Scheme 1.Recently, we developed a promising class of steroid compounds, which are derivatives of 3,20-dihydroxy-19-norpregn-1,3,5(10)-trienes of the natural 13β-and epimeric 13α-series (Scheme 2), and they exhibit high cytotoxicity against the hormone-dependent MCF7 breast cancer cell line and low toxicity against normal MCF-10A breast epithelial cells (Kuznetsov et al., 2018a;Kuznetsov et al., 2018b).

Chemistry
The compounds under study were synthesized, among others in the series, from the commercially available natural hormone estrone via a multistage chemical synthesis, including etherification of estrone, epimerization of the 13-center of the steroid core (in 13α-series), building and modification of the reactive fragment at the 16-and 17-positions of the steroid, and final reduction of the ketone group and cleavage of the steroid methyl ether.Diastereoisomeric products, if they were obtained, were separated by column chromatography on silica gel.The structures and purity of the obtained compounds were confirmed by physicochemical methods (Kuznetsov et al., 2018a;Kuznetsov et al., 2018b).
Briefly, the cells were seeded in 24-well plates (Corning, New York, USA) in 900 μL of standard medium.Compounds at different concentrations in 100 μL of the appropriate medium were added, and the cells were grown for 72 h.After incubation with the compounds, the medium was removed, the MTT reagent that was dissolved in the medium was added to the final concentration of 0.2 mg/mL to each well, and the incubation was performed for 2 h.Then, the cell supernatants were removed, and purple formazan crystals were dissolved in DMSO (350 μL per well).The plates were gently shaken, and the absorbance was measured at 571 nm with a reference wavelength of 630 nm on a MultiScan reader (ThermoFisher, Waltham, MA USA).The viability of the cells was expressed as a percentage of the control.The combination index (CI) was calculated as described previously (Miladiyah et al., 2020;Zhao, Au, Wientjes, 2010).

Cell cycle analysis
MDA-MB-231 breast cancer cells were seeded in 6-well plates and treated with the compounds for 48 h.Cell sediments were treated with a PI buffer containing 50 μg/mL propidium iodide (PI), 100 μg/mL RNAse A (Sigma-Aldrich, Burlington, MA, USA), 0.1% sodium citrate, and 0.3% NP-40 (Helicon, Moscow, Russia) for 30 min in the dark.Cell cycle data were acquired by measuring the DNA content using a Cytoflex flow cytometer 26 (Beckman Coulter Brea, California, USA) in the PerCP-A channel.Data analysis was performed with CytExpert (Beckman Coulter).
Immunoblotting MDA-MB-231 cells were washed twice in phosphate buffered saline and incubated for 10 min on ice in total lysis buffer containing 50 mM Tris-HCl at pH 7.4, 1% SDS, 1% Igepal CA-630, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 µg/mL each of aprotinin, leupeptin, pepstatin, 1 mM Na-orthovanadate, and 1 mM NaF (Sigma-Aldrich, Burlington, MA, USA).The samples were sonicated five times for 5 s each at 30% output and centrifuged for 5 min at 15 000 × g, and supernatants were then used as total cell extracts.Total protein content was determined by the Bradford method.
MDA-MB-231 cell lysates were separated in 10% SDS-PAGE under reducing conditions, transferred to a nitrocellulose membrane (GE HealthCare, Chicago, Illinois, USA), and processed according to a standard protocol.GLUT1, CDK 4/6, Cyclin D1, full and cleaved PARP, BAD and Bcl-2 antibodies were obtained from Cell Signaling Technology; the antibodies against α-tubulin (Cell Signaling Technology) were added to standardize loading.Goat anti-rabbit IgGs (Jackson ImmunoResearch, Ely, Cambridgeshire, United Kingdom) conjugated to horseradish peroxidase were used as secondary antibodies.Signals were detected using the ECL reagent, as described in Mruk and Cheng's protocol (Mruk, Cheng, 2011), and an ImageQuant LAS4000 system (GE HealthCare, Chicago, Illinois, USA).

Antiproliferative activity of the 13α-/β-steroids
To assess the antiproliferative activities of the synthesized compounds, a cell line of triple-negative breast cancer MDA-MB-231 was selected.The MDA-MB-231 cells were seeded on a plate and after 24 h were treated with compounds in various doses.As shown in Figure 2, compound kuz8b at micromolar concentrations did not considerably affect the growth of MDA-MB-231 cells, whereas its 13β-analog kuz7 significantly suppressed cell growth.However, 17.6 μM kuz8b causes 50% inhibition of the growth of MDA-MB-231 cells (Table I).The IC 50 value of kuz7 was slightly less than 5 μM.Cisplatin and 5-fluorouracil were used as reference drugs.As indicated in Table I, drugs had various effects on MDA-MB-231 cells.Cisplatin inhibited the growth of MDA-MB-231 cells with an IC 50 of 12.7 µM, whereas 5-fluorouracil exhibited weak activity with an IC 50 greater than 25 µM.Thus, compound kuz7 was more active than cisplatin and fluorouracil; compound kuz8b was inferior in activity only to cisplatin.
Figure 3 shows the distribution of the MDA-MB-231 cell line within the cell cycle after incubation with kuz7 for 48 h.The number of apoptotic cells (the subG1 phase of the cell cycle) significantly increased as the concentration of the active compound increased: 8% apoptotic cells of the control cells population versus 62% apoptosis level in the sample treated with 10 µM steroid kuz7.Moreover, the tested compound kuz7 caused a considerable reduction of the G0/G1 phase population of cells treated with 1.2-10 µM of kuz7 simultaneously with a gradual decrease in the S phase of the cell cycle.Of note, the percentage of cells in the G2/M phase did not change in the range of low concentrations and was reduced due to high-concentration treatment with kuz7.Thus, the effect of kuz7 on the cell cycle of MDA-MB-231 cells is characterized by a significant increase in the number of apoptotic cells with a simultaneous reduction of the G0/G1 and S phase of cells as the compound concentration increases.

Cell cycle analysis of MDA-MB-231 treated with 13α-/β-steroids
We used the TNBC cell line MDA-MB-231 to investigate the effects of kuz7 and kuz8b on the cell cycle.
The analysis of the distribution of MDA-MD-231 cells in populations, according to the DNA content after treatment with kuz8b, is shown in Figure 4. Here, we identified similar patterns characteristic of the steroid kuz7 from the 13ß-series.Compound kuz8b was tested in the range of concentrations from 1.2 to 20 µM.Of note, we used a higher concentration of kuz8b because it had a slightly lower cytotoxic effect than the previously described kuz7.We demonstrated that an increase in the effective concentration of kuz8b caused the accumulation of a fraction of apoptotic MDA-MB-231 cells.The reduction of the MDA-MB-231 cell population by half was achieved after treatment with 20 µM kuz8b for 48 h.In addition to the increase in apoptosis, there was a decrease in the percentage of cells in the G0/G1 phase: 17.6% in 20 µM kuz8b-treated cells vs. 52.6% in control cells.Unlike kuz7, there was no significant change in cell distribution within the S and G2/M phases of the cell cycle in the range of tested concentrations for kuz8b.Thus, kuz8b can be characterized by two main trends of action in MDA-MB-231 cells involving a cytotoxic effect: an increase in the apoptotic fraction and a parallel reduction in the percentage of the G0/G1 phase of the cell cycle.

Signaling pathways regulated by 13α-/β-steroids
In subsequent experiments, we analyzed the signaling pathways that are regulated by the synthesized compounds.MDA-MB-231 cells were treated with compounds, and then, the expression of signaling proteins was determined by immunoblotting.The data obtained are shown in Figure 5. CDK4/6 and cyclin D1 are the main regulators of the cell cycle.In MDA-MB-231 cells treated with the compounds, we found no significant changes in the expression of these proteins.A slight decrease in Considering that kuz7 is more active than kuz8b, we conducted further experiments with kuz7 alone.It is known that inhibition of glucose transport can lead to the activation of oxidative phosphorylation (OxPhos).The application of kuz7 in combination with a respiratory chain inhibitor may provide significant antiproliferative effects against MDA-MB-231 cells.As the OxPhos inhibitor, we chose metformin (MF), which is used to treat diabetes mellitus.The key target of metformin is complex I of the mitochondrial respiratory chain (Feng et al., 2022).
FIGURE 5 -Signaling pathways regulated by 13α-/β-steroids.MDA-MB-231 cells were treated with 0, 1.2, 5, and 10 μM of tested compounds.The GLUT1, CDK 4/6, and cyclin D1 expressions were detected by immunoblotting.α-Tubulin was used as a loading control.CDK4 expression was observed for cells treated with the steroid kuz7.Glucose supply is very important for MDA-MB-231 breast cancer cells, and the growth of such cancer depends on the availability and transport of glucose.
Glucose enters cells through its transporters, and GLUT1 is one of the proteins responsible for glucose uptake.We found that the steroid kuz7 reduces the expression of GLUT1, as shown in Figure 5.As shown in Figure 6, kuz7 caused approximately 40% inhibition of cell growth, and the effects of MF were weak when it was used alone.Using the agents in combination resulted in significant suppression of MDA-MB-231 cell growth.
We hypothesized that the chosen combination of compounds would provide an effect on apoptosis proteins.The results of the experiment revealed that compound kuz7 alone causes an increase in the level of cleavage of poly(ADP-ribose) polymerase (PARP) (Figure 7), which is a biomarker of induced apoptosis (Moghtaderi et al., 2018;Saraste, Pulkki, 2000).MF treatment did not result in PARP cleavage compared to that in control samples.The combination treatment induced PARP cleavage as high as that in MDA-MB-231 cells treated with kuz7 alone.Among the factors that restrain programmed cell death are the disbalance of Bcl-2/BAD proteins, reduced caspase function, and disrupted death receptor signaling (Elmore, 2007).The expression of pro-apoptotic protein BAD was high and did not change after incubation of cells with kuz7, MF, or their combination.The expression of the anti-apoptotic protein Bcl-2 did not change when MDA-MB-231 cells were treated with kuz7 or MF, whereas the drug combination significantly reduced the expression of this factor.Thus, the combination of kuz7 with MF has a significant antiproliferative effect on MDA-MB-231 cells and induces apoptosis, including through a decrease in Bcl-2 expression.

DISCUSSION
TNBC is the most aggressive type of breast cancer and accounts for 15-25% of all breast tumors; it is associated with a poor prognosis of the course of the disease (da Silva et al., 2020;Medina et al., 2020).In many cases, this type of cancer is resistant to treatment; hence, the investigation of new compounds and the development of new therapeutic approaches targeted to TNBC treatment is a promising area of research (Moghtaderi et al., 2018;Nikolić et al., 2015;Won, Spruck, 2020).Currently, novel compounds (including those in the steroid series) are being actively designed and studied by scientists around the world (Fröhlich et al., 2018;Kovačević et al., 2016;Moghtaderi et al., 2018;Nikolić et al., 2018;Won, Spruck, 2020).Scheme 1 demonstrates some of the natural and synthesized compounds exhibiting anticancer activity.
Cucurbitacins are a class of highly oxidized tetracyclic triterpenes present in several plants used in traditional Chinese medicine treatments.Analysis of the antiproliferative properties of cucurbitacins was performed by several research groups (Alghasham, 2013;Garg, Kaul, Wadhwa, 2018;Kong et al., 2014, Lan et al., 2013).The first significant study of cucurbitacin E (CuE, Scheme 1, Compound 1) on TNBC cells was described previously (Lan et al., 2013).CuE inhibits the growth of MDA-MB-231 breast cancer cells in a dose and timedependent manner.Further analysis showed that CuE induces G2/M phase arrest and apoptosis.CuE induced the cleavage of caspase 3 and upregulated p21 and p27.CuE enhanced the antiproliferative potency of cisplatin (Lan et al., 2013).In another study, Yanjie Kong et al. confirmed that CuE significantly inhibited the growth of TNBC cells, leading to the arrest in the G2/M phase of the cell cycle (Kong et al., 2014).CuE treatment resulted in a decrease in the number of EdU-positive proliferating cells in a dose-dependent manner and further apoptosis within 24 h.The described compound showed a strong growth-inhibiting effect on five TNBC cell lines with IC 50 values from 10 to 70 nM.CuE in concentrations from 100 to 200 nM reduced the expression of proteins such as cyclin D1 (cell cycle regulator), survivin, Mcl-1, XIAP, and Bcl-2 (anti-apoptotic proteins) (Kong et al., 2014).Thus, CuE has a higher antiproliferative potency than that of the obtained steroids kuz7 and kuz8b and reference compounds in our study.CuE induced G2/M cell cycle arrest, whereas the tested steroids increased the subG1 cell population, decreased the G0/G1 and S phase cell population at lower doses and only decreased G2/M cell population at higher doses.CuE alone and steroid kuz7 in combination with MF were able to reduce Bcl-2 expression in cancer cells.
Evgenija Djurendić et al. evaluated the effect of D-seco and D-homo androstane derivatives on TNBC cells.Compounds 2 (an IC 50 of 10 µM) and 3 (an IC 50 of 9 µM) showed moderate antiproliferative activity against MDA-MB-231 cells and a low cytotoxic effect on the estrogen-positive cell line MCF7 (IC 50 >100 µM) (Djurendić et al., 2009).Strahinja Kovačević et al. used a method of quantitative structure-activity relationship (QSAR) to predict the antiproliferative activity against breast cancer cells of A-and B-modified D-homo lactone and D-seco androstane derivatives (Kovačević et al., 2016).In the first group, there were compounds with D-homolactone function in the steroid nucleus, while in the second group, there were steroids with D-seco function.Eventually, the studied compounds showed antiproliferative activity against MDA-MB-231 with IC 50 values from 10 µM and higher (Kovačević et al., 2016).
Andrea Nikolić et al. investigated the antitumor activity of novel 6-substituted 4-en-3-one D-secosteroidal dinitriles.Among them, compound 5 showed a good cytotoxic effect on the MDA-MB-231 cell line with an IC 50 value of 2.8 µM, and early and late apoptosis was also observed by different methods.Moreover, the described compounds did not affect the proliferation of normal MRC-5 cells (Nikolić et al., 2018).The same group reported the antitumor potential of steroid 16,17-seco-16,17a-dinitriles.Compound 6, the 3-keto-1,4-diene derivative, has been shown as a selective agent with antiproliferative activity against estrogen-dependent (MCF7) and estrogen-independent (MDA-MB-231) breast cancer cell lines at submicromolar concentrations with IC 50 values of 0.52 and 0.11 µM, respectively (Nikolić et al., 2015).Novel diastereomeric 16-hydroxymethyl-19-nortestosterone derivatives prepared by Birch reduction from the corresponding 3-methoxy-16hydroxymethylestra-1,3,5(10)-trien-17-ol isomers with known configurations were described by Gyula Schneider (Schneider et al., 2016).One of the obtained compounds, 17α-p-methoxybenzoyloxy19-nortestosterone estra-4en-3-one (Scheme 1, compound 7), at a concentration of 30 µM exhibited 71% growth inhibition of MDA-MB-231 cells and was as active as the chemotherapy drug cisplatin (Schneider et al., 2016).Thus, the IC 50 values of steroids and steroid-like compounds against TNBC vary considerably.Usually, these compounds inhibit cancer cell growth at doses of 1 µM and slightly higher, except for compounds with very high activity, such as CuE.The antiproliferative potency of the 13β-steroid kuz7 is similar to that described for several lead compounds, whereas the 13α-steroid 8b is less active.
Compounds isolated from natural products can provide hormonal and antihormonal effects, mimicking the action of hormones.Such compounds are also considered potent agents that inhibit the growth of triplenegative breast cancers.The activity of the phytoestrogen apigenin (compound 8, Scheme 1) has been described in several recent papers (Bauer et al., 2020;Lee et al., 2019;Nasir et al., 2020).We demonstrated the activity of apigenin against various cancer cell lines (Scherbakov, Andreeva, 2015).Cultivation of HER2-positive breast cancer SK-BR-3 (HTB-30) cells in the presence of apigenin resulted in a decrease in HER2/neu expression, accompanied by cleavage of an apoptosis substrate PARP.The search for novel combinations of steroidlike agents to inhibit cancer is actively being conducted.For instance, Hassan Moghtaderi et al. showed that the combination of gallic acid and curcumin (compound 9, Scheme 1) strongly decreased MDA-MB-231 cell growth (Moghtaderi et al., 2018).Moreover, the applied combination increased the ROS level and cytotoxic activity along with the glutathione depletion in MDA-MB-231 cells.Vivek Kumar Soni et al. described that metabolite transporters and receptors (GLUT1, MCT-1, MCT-4, and HCAR-1) were upregulated in high glucoseexposed HepG2 cells (Soni et al., 2021).Curcumin 9 inhibited the elevated expression of these enzymes, transporters, and receptors in tumor cells.

CONCLUSIONS
This investigation was performed to explore the effect of 13α-and 13β-steroids on the growth of triplenegative breast cancer cells.The antiproliferative activities of the obtained compounds kuz8b and kuz7 against MDA-MB-231 cells were evaluated.The 13β-steroid kuz7 selected for study was more active against MDA-MB-231 than the 13α-steroid kuz8b.Analysis of the cell cycle revealed common patterns regarding the effects of both steroids.After treatment with the compounds, MDA-MB-231 cells were in the subG1 phase, which Alexander M. Scherbakov, Yury V. Kuznetsov, Margarita A. Yastrebova, Alvina I. Khamidullina, Danila V. Sorokin, Maria O. Tserfas, Inna S. Levina indicates the induction of apoptosis.Compound kuz7 at micromolar concentrations reduced the expression of GLUT1, a transmembrane protein responsible for the facilitated diffusion of glucose across a cell membrane.The combination of kuz7 with biguanide MF was shown to be synergistic.Taking into account the results of our experiments, including the high activity of kuz7 against triple-negative cancer cells, the detected induction of apoptosis, and decrease in GLUT1 expression, the 13β-steroid kuz7 is of interest for further preclinical studies, alone or/and in combination with the complex I inhibitor MF.

FIGURE 2 -
FIGURE 2 -Antiproliferative activity of compounds kuz7 and kuz8b against MDA-MB-231 cells (the cell viability was assessed by MTT assays after 72 h of growth with steroids).

FIGURE 3 -
FIGURE 3 -Analysis of the cell cycle of MDA-MB-231 after incubation with compound kuz7 for 48 h.

FIGURE-
FIGURE -Analysis of the cell cycle of MDA-MB-231 after incubation with compound kuz8b for 48 h.

FIGURE 6 -
FIGURE 6 -Combinatorial synergism of kuz7 and metformin (MF).MDA-MB-231 cells were exposed to the combination of kuz7 and MF or each treatment alone for 72 h, and cell viability was assessed using MTT assay (the combination index was <0.8, when the concentration ratio was 800).Error bars in the figure represent mean ± SD.P<0.05: * -versus vehicle control and each drug alone.

TABLE I -
IC 50 values of compounds kuz7 and