Red Clover (Trifolium pratense) and Cancer (anticancer research): evidence and sources

To review beneficial effects of red clover (Trifolium pratense L.) on biological processes. A systematic review was carried out according tothe PRISMA checklist. Using MeSH keywords, 385 articles were extracted from national and international databases (PubMed, Science Direct, SID, and Google Scholar search engine) without time limit. After removing duplicates, 80 articles were reviewed. Our findings revealed that the compositions of red clover promote anti-oxidant and anti-cancer effects. Furthermore, they exude beneficial effects on cardiovascular functionand improvesymptoms of menopause. Also, these compounds can regulate blood glucose and lipid markers. The effects of the herb on have also been investigated on various tissues including endometrium, breast, skin and reproductive system. In recent studies, the anti-cancer effects of clover (Trifolium) extract co-administrated with standard drugs have been reportedin different cancers.

Triple-negative breast cancer (TNBC) presents significant therapeutic challenges. This study investigates the combination effects of Trifolium pratense L. (red clover) and doxorubicin (DOX) on the Wnt/β-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and apoptosis in 4T1 tumor-bearing BALB/c mice. Female BALB/c mice were divided into six (n=10) groups: control, DOX (5 mg/kg), and three treatment groups receiving 100, 200, or 400 mg/kgextract alongside DOX, and a single dose of 400 mg/kg. Tumor size was measured using Vernier calipers, and survival rates were analyzed through Kaplan-Meier curves. Tumors were removed to analyze histological examinations and gene expression of,,,,,,, and. Immunohistochemical staining was performed to evaluate p53, Ki-67, β-catenin, Cdh1, and vimentin expression. Co-treatment of(400 mg/kg) with DOX (5 mg/kg) synergistically reduced cell proliferation and increased apoptosis by increasing p53 and decreasing Ki-67 expression in a dose-dependent manner. This co-treatment effectively inhibited the Wnt/β-catenin pathway by upregulating antagonists (and), modulating β-catenin accumulation, and reversing EMT through increased E-cadherin expression and decreased vimentin (protein level) and(gene expression) levels. extract shows potential as an adjuvant therapy against TNBC by targeting the Wnt/β-catenin pathway and reversing EMT while enhancing DOX efficacy. Further research is warranted to explore additional anticancer mechanisms ofextract.

B-cell acute lymphoblastic leukemia (B-ALL), a prevalent malignancy predominantly affecting children, poses challenges such as drug resistance and cytotoxicity despite available treatment methods. The persistence of these challenges underscores the necessity for innovative therapeutic approaches to enhance efficacy. Natural compounds derived from plants, recognized for their potential to inhibit cancer cell growth, have drawn attention. Trifolium pratense extract, known for its significant anticancer properties in previous studies, was the focus of this investigation. This experimental study aimed to explore the impact of T. pratense extract on apoptosis and autophagy in NALM-6 cells. The cells were exposed to varying concentrations of the extract at specific time intervals, with viability and metabolic activity assessed using Trypan blue exclusion and MTT assays. Flow cytometry was employed to evaluate apoptosis using Annexin V/PI staining and ROS production using DCFH-DA staining. Real-time PCR was used to quantify gene expression related to apoptosis, autophagy, and oxidative stress, with data analysis performed using GraphPad PRISM software. Trifolium pratense extract demonstrated the capacity to induce apoptosis, autophagy, and significantly increase ROS production in NALM-6 cells. These effects were facilitated by the upregulation of corresponding genes. The MTT assay revealed an IC50 of 231 μg/mL at 48 h, and Flow cytometry analysis showed a 51.8% increase in apoptosis in this cell line. Overall, this study emphasizes the effectiveness of T. pratense extract in inducing autophagy and apoptosis pathways in NALM-6 cells derived from B-cell acute lymphoblastic leukemia, suggesting its potential as a candidate for further investigation as a supplement in ALL treatment.

Glioblastoma (GBM) is the most common type of primary brain tumors in adults, characterized by its ability to proliferate rapidly and its tendency to aggressively and strongly invaded the surrounding brain tissue. The standard treatment approach of GBM is surgical resection followed by simultaneous chemotherapy and radiation. However, a significant number of GBM cases develop resistance to currently used chemotherapeutic drugs. Therefore, there is a need for the development of new chemotherapeutic agents.pL. is an endemic plant containing various isoflavones such as biochanin A, genistein, daidzein, and formononetin in high concentrations, and it has been shown in various studies that these molecules can function as anticancer agents. The present study was designed to determine the effect of the possible anticarcinogenic effects of thepL. which grown in our country and to obtain new treatment approaches alternative to the classical treatment protocols applied in the treatment of GBM. C6 glioblastoma cells were cultured withpL. Cell proliferation, apoptotic cell morphology, and cell structure were evaluated with CCK8, Annexin V, cytochrome c, CD117, and Betatubulin labeling, respectively. And also, investigated effects of this Turkish tetraploid on GBM by TEM. Decreased cell proliferation and increased number of apoptotic cells were observed depending on the increasing doses ofpL. In addition, intense morphological changes were detected depending on increasing doses. In this context, we believe that the plantpL., may be a new alternative and adjuvant agent for the treatment of GBM.

Abstract Trifolium pratense L. (Red clover— T. pratense ) commonly consumed as a healthy beverage has been demonstrated to have various biological activities including antioxidant and anticancer effects. The aim of this study was to investigate the antimetastasis effects of doxorubicin (DOX) and T. pratense extract in 4T1 tumor‐bearing BALB/c mice. In this study, 56 female BALB/c mice were randomly divided into seven groups ( n = 8/group) to receive DOX and T. pratense extract in three different doses (100, 200, and 400 mg/kg/day) for 35 days. On day 36 after starting treatments, serum cytokines (IL‐8 and IL‐6) were measured. Immunohistochemical (IHC) staining was performed for GATA‐3 in the brain and lung, and for CK5/6 in tumor tissues. Metastasis‐related gene (matrix metalloproteinase‐2 [ MMP‐2 ] and sirtuin‐1 [ SIRT‐1 ]) expressions were also measured by real ‐ time PCR. Our results showed that cotreatment with DOX and T. pratense extract improved stereological parameters (i.e., reduction in the volume of metastatic tumors) in the lung and brain and decreased the serum levels of inflammatory cytokines (IL‐8 and IL‐6). DOX and T. pratense extract synergistically down‐regulated MMP‐2 and up‐regulated SIRT‐1 genes, decreased the number of CK5/6‐positive cells in tumor tissues, and inhibited metastasis of GATA‐3‐positive cells into the lung and brain. The combination of T. pratense extract and DOX synergistically inhibited the metastasis of 4T1 xenograft cells in a dose‐dependent manner.

Abstract Therapeutic strategies against triple‐negative breast cancer (TNBC) are associated with drug‐induced toxicities. The tropical edible red clover ( Trifolium pratense L.) is rich in polyphenolic compounds which confer the plant potential anticancer properties. The aim of this study was to investigate the effects of T. pratense and doxorubicin (DOX) on the apoptosis and proliferation of 4T1 tumor cells in an allograft model of tumor‐bearing BALB/c mice. Fifty‐six female 4T1‐tumor bearing‐ BALB/c mice were randomly divided into 7 groups ( n = 8/group) to receive different doses and combinations of DOX and T. pratense extract for 35 days. On the 36th day, serum estradiol (E2), IL‐12 and IFN‐γ cytokines, and glutathione peroxidase (GPx) activity were measured. Tumor's ferric reducing antioxidant power (FRAP) and the expressions of apoptosis‐related genes (p53, Bax, Bcl‐2, and caspase‐3) were also evaluated. Immunohistochemical staining for Ki‐67 and p53 were performed. Our results showed that the co‐treatment of DOX and T. pratense (100–400 mg/kg) inhibited the proliferation of 4T1 tumor cells in dose‐ and time‐dependent manners. The co‐treatment of DOX and T. pratense (especially at the dose of 400 mg/kg) decreased the serum level of E2 (as a stimulant for breast tumor growth) and increased the serum levels of IL‐12 and IFN‐γ along with significant increments in serum GPx and tumor FRAP activities. The co‐administration of DOX and T. pratense also decreased the expression of Ki‐67 proliferation marker and increased the number p53 positive (i.e., apoptotic) cells within tumors. This was accompanied with the upregulation of pro‐apoptotic and down‐regulation of antiapoptotic genes. The key findings indicated the synergistic effects of DOX and T. pratense against TNBC xenografts.

Uncontrolled growth and migration and invasion abilities are common for cancer cells in malignant tumors with low therapeutic effectiveness and high mortality and morbidity. Estrogen receptor β (ERβ), as a member of the nuclear receptor superfamily, shows potent tumor suppressive activities in many cancers. Phytoestrogens' structural resemblance to 17 β-estradiol allows their binding to ERβ isoform predominantly, and therefore, expression of genes connected with elevated proliferation, motility and invasiveness of cancer cells may be downregulated. Among polyphenolic compounds with phytoestrogenic activity, there are isoflavones fromL. (red clover) sprouts, containing high amounts of formononetin and biochanin A and their glycosides. To determine the source of the most biologically active isoflavones, we obtained four extracts from sprouts before and after their lactic fermentation and/or β-glucosidase treatment. Our previous results of ITC (isothermal titration calorimetry) modelling and a docking simulation showed clover isoflavones' affinity to ERβ binding, which may downregulate cancer cell proliferation and migration. Thus, the biological activity ofsprouts' extracts was checked under in vitro conditions against highly invasive human breast cancer cell line MDA-MB-231 and non-invasive human breast cancer cell line MCF-7 cells. To compare extracts' activities acquired for cancer cells with those activities against normal cells, as a third model we choose human umbilical vein endothelial cells (HUVEC), which, due to their migration abilities, are involved in blood vessel formation. Extracts obtained from fermented sprouts at ICdosages were able to inhibit migration of breast cancer cells through their influence on intracellular ROS generation; membrane stiffening; adhesion; regulation of MMP-9, N-cadherin and E-cadherin at transcriptional level; or VEGF secretion. Simultaneously, isolated phenolics revealed no toxicity against normal HUVEC cells. In the manuscript, we proposed a preliminary mechanism accounting for the in vitro activity ofL. isoflavones. In this manner,sprouts, especially after their lactic fermentation, can be considered a potent source of biological active phytoestrogens and a dietary supplement with anti-cancer and anti-invasion properties.

Breast cancer is currently the most common cancer in women worldwide. Tamoxifen is used for the treatment of both early and advanced estrogen receptor positive breast cancer. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. The effect of T. pratense extract on breast cell lines (MCF-7 and MDA-MB-231) was investigated here. The cells were treated with T. pratense extract and cell viability was evaluated using trypan blue staining, MTT assay and lactate dehydrogenase activity measurement. Apoptosis, autophagy cell death was detected using fluorescent dye staining. Griess assay was performed to nitric oxide (No) measurement. Change in expression level of some apoptotic and autophagic- related genes was detected using real-time PCR. The combination effect of exteract and tamoxifen was evaluated by calculating the combination index and dose reduction index (DRI) values. T. pratense extract reduced the cell viability in dose and time- dependent manner. Apoptosis and autophagy was increased after treatment with T. pratense extract. Nitric oxide production significantly reduced in cells and combination of tamoxifen and T. pratense extract had a synergistic cytotoxic effect on breast cancer cell lines. T. pratense as a dietary supplement can be beneficial for some patients with breast cancer who receive conventional chemotherapy.