Unveiling the potential anti‐cancer activity of calycosin against multivarious cancers with molecular insights: A promising frontier in cancer research

Abstract Background Calycosin may be a potential candidate regarding chemotherapeutic agent, because already some studies against multivarious cancer have been made with this natural compound. Aim This review elucidated a brief overview of previous studies on calycosin potential effects on various cancers and its potential mechanism of action. Methodology Data retrieved by systematic searches of Google Scholar, PubMed, Science Direct, Web of Science, and Scopus by using keywords including calycosin, cancer types, anti‐cancer mechanism, synergistic, and pharmacokinetic and commonly used tools are BioRender, ChemDraw Professional 16.0, and ADMETlab 2.0. Results Based on our review, calycosin is available in nature and effective against around 15 different types of cancer. Generally, the anti‐cancer mechanism of this compound is mediated through a variety of processes, including regulation of apoptotic pathways, cell cycle, angiogenesis and metastasis, oncogenes, enzymatic pathways, and signal transduction process. These study conducted in various study models, including in silico, in vitro, preclinical and clinical models. The molecular framework behind the anti‐cancer effect is targeting some oncogenic and therapeutic proteins and multiple signaling cascades. Therapies based on nano‐formulated calycosin may make excellent nanocarriers for the delivery of this compound to targeted tissue as well as particular organ. This natural compound becomes very effective when combined with other natural compounds and some standard drugs. Moreover, proper use of this compound can reverse resistance to existing anti‐cancer drugs through a variety of strategies. Calycosin showed better pharmacokinetic properties with less toxicity in human bodies. Conclusion Calycosin exhibits excellent potential as a therapeutic drug against several cancer types and should be consumed until standard chemotherapeutics are available in pharma markets.


| INTRODUCTION
Cancer remains one of the most formidable challenges to human health, with its devastating impact reaching every corner of the globe. 1 The severity of cancer seems more than any other disease, including antibiotic resistance and the coronavirus disease 2019 (COVID-19). 2 According to the National Cancer Institute (NCI), globally, people are affected by multiple forms of cancer. 3,4ome conventional therapies include surgical resection, chemo-radiotherapies, adjuvant chemotherapies, neoadjuvant therapy, hormonal therapies, monoclonal antibodies, immunotherapies, nanomedicines, and small molecular inhibitors are recommended by oncologists. 5,6Despite significant advancements in the understanding and treatment of various malignancies, the quest for novel and effective anti-cancer agents continues unabated due to having some common drawbacks, like bearing some potential side effects and toxicities. 7herefore, introducing natural compounds derived from plants has long been a source of inspiration for drug discovery due to their rich chemical diversity, historical use in traditional medicine, and reliable therapeutic option for treating many forms of human cancer [8][9][10][11][12] Currently, medicinal plants or their derivatives account for about 70% of the anti-cancer compounds, thus, playing the lead role in developing anti-cancer drugs. 13,14Because these compounds are safe, less-toxic, cost-effective, and readily available sources from villages to cities and underdeveloped to developed countries. 15][18][19][20] Calycosin, a functional phytoestrogen belonging to the isoflavone category, can be found in the root of Astragalus membranaceus. 21Calycosin has been traditionally tested in herbal medicine for its myriad of health benefits, including anti-inflammatory, 22 anti-microbial, 23 neuroprotective, 24 and anti-diabetic. 25These pharmacological activities of calycosin are due to agonist and antagonist properties with multiple receptors. 26However, it is in the realm of oncology that this compound is emerging as a promising candidate for novel anti-cancer therapies.Some experimental studies of calycosin in numerous study models, including in silico, in vitro, preclinical, and clinical trials against multiple cancer types, have been done. 27t has gained attention for its potential anti-cancer activity due to its ability to influence various cellular processes that play a role in cancer development and progression, including cell cycle arrest, apoptosis induction, inhibition of angiogenesis, anti-inflammatory effects, metastasis suppression, and hormone receptor modulation: inhibition of signaling pathways and showing antioxidant properties, to prevent the onset and spread of cancer.Additionally, calycosin may work with some other natural compounds and conventional chemotherapy drugs to enhance the activity of these drugs and alleviate the resistance profile of existing drugs. 28Calycosin's natural availability, creation of nanocarriers to boost bioavailability, pharmacokinetic profile, and low toxicity make it a popular choice for cancer treatment.As far as we know, although some experimental studies have already been conducted, a detailed overview regarding the onco-therapeutics potential of calycosin with molecular in treating multivarious cancers has not yet been revealed.
Therefore, the present review aims to shed light on the multifaceted anti-cancer activities of calycosin and explore the underlying molecular mechanisms, thereby emphasizing its potential significance in cancer treatment, which inspire biological researchers to develop new, effective, clinically proved drugs to combat human malignancies.

| Black cohosh (Cimicifuga racemosa)
Black cohosh contains calycosin and has been investigated for its potential to inhibit the growth of breast cancer cells.It may also help alleviate some cancer treatment-related symptoms. 40A B L E 1 Reported plant sources of calycosin.Licorice root contains calycosin and has been studied for its anti-inflammatory and potential anti-cancer effects.

Thermopsis lanceolata
It may be used as part of a broader approach to cancer treatment. 43 2.9 | White willow bark (Salix alba) White willow bark contains calycosin and has been researched for its anti-inflammatory properties.It may be used to manage inflammation associated with cancer including human colon and lung cancer cells.This traditional Chinese herb contains calycosin and has been explored for its potential in cancer treatment, particularly in combination with other herbs.The chemical structure of calycosin is depicted at Figure 1.

| THE EXTRACTION, PURIFICATION, AND QUALITY INSPECTION METHODS OF CALYCOSIN
The information related to the extraction, purification, and quality inspection methods of calycosin is very rare.
The study focuses on efficiently extracting and purifying calycosin, a bioactive substance found in Astragali Radix.Calycosin-7-glucoside can degrade to calycosin.The optimal extraction conditions for calycosin were determined using single-factor and orthogonal studies.The requirements involved using 100% ethanol and 2.5 mol/L hydrochloric acid in a solid-to-liquid ratio of 1:40, followed by a 2-h compound extraction.The calycosin was purified using a two-phase solvent system of n-hexane, ethyl acetate, ethanol, and water (3:5:3:5, v/v).This process yielded 1.3 mg of calycosin with a purity of 95.8% and a recovery rate of 85.9% from 264.9 mg of crude extraction.Purifying calycosin involves isolating it from the plant material and other impurities.Various analytical methods, such as HPLC, melting points, UV, FTIR, ESI-MS, 1 H NMR, and 13 C NMR spectra, were employed to ascertain the structure of calycosin.Hydrolysis with hydrochloric acid is necessary to convert calycosin glycosides into their active state.HSCCC (high-speed countercurrent chromatography) achieves a high level of purification.Regarding to quality inspections of calycosin, especially in the context of herbal medicine or dietary supplements, various analytical methods including high-performance liquid chromatography (HPLC), thinlayer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, ultraviolet-visible (UV-VIS) spectroscopy, mass spectrometry (MS), elemental analysis, microscopic examination, HPLC fingerprints, quantitative analysis, residue analysis, heavy metal analysis, microbiological testing, and stability testing can be done. 46I G U R E 1 Chemical structure of calycosin.

PHARMACOLOGY AND BASIS OF ANTI-CANCER ACTIVITY BY CALYCOSIN
Natural phytochemicals or conventional drugs can target a specific receptor to either increase or reduce a particular cell function. 47Regarding managing cancerous tissues, phytochemicals must show specific activities by interacting with receptors.Consequently, Chen et al. reported that calycosin mediates anti-cancer properties, including inhibiting growth and inducing apoptosis by targeting estrogen receptor beta (ER-β) in numerous breast cancer cells by suppressing IGF-1R, combined with the selective modulation of MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. 48Calycosin suppressed the proliferation of ER-positive and negative breast cancer cells through acting WDR7-7-GPR30 signaling via reducing phosphorylation of therapeutic targeting protein including SRC, EGFR, ERK1/2, and Akt in a dose-dependent manner. 49Calycosin exerted anti-cancer activity in vitro model IGF-1R mediated cell proliferation by controlling PI3K/Akt signaling pathways and of calycosin-mediated apoptosis with inhibiting cell invasion by targeting SIRT1 activator which induces activation of AMPK-induced inhibition of Akt/mTOR axis in HT29 cell line. 50Moreover, this molecule could abolish transforming growth factor ß-(TGF-β-) induced epithelial-to-mesenchymal transition via altering the Wnt mechanism. 51Calycosin interacts with EGFR and thus behaves as an antagonist, downregulating EGFR at in vitro studies with Mice SKBR3 cells and MCF-7 cells. 49The summary of the molecular interaction of calycosin with numerous receptors leading mediate anti-cancer activities is tabulated in Table 2.

| EVIDENCE OF ANTI-CANCER
PERSPECTIVES REGARDING CALYCOSIN

| In silico anti-cancer activity of calycosin
In silico is an early invention process that entails identifying biomarkers, disease progression, and potential treatments for various illnesses.This procedure offers crucial insights into the molecular processes that create and spread cancerous cells in the event of cancer. 52eng et al. reported that apoptosis is a significant factor in biological cell death, where calycosin upregulates Bax, caspase 3, and cleaved caspase 3 and downregulates the expression of Bcl2, determined by GO and KEGG enrichment analyses. 53 The second most frequent cancer-related death among women is breast cancer.Previous scientific studies supported that calycosin may be practical therapeutic activity against breast cancer in different study models.It mediated anti-carcinogenic activity through restrictions on the proliferation of cells and tumor cell growth via upregulating estrogen receptor beta (ER-β) and downregulating of 1R insulin-like growth factor 1 (IGF-1) in MCF-7 and T-47D cell in a dose-dependent way. 58n treating human breast cancer cells, calycosin (0.5-100 μM) diminished Akt phosphorylation and reduced PI3K/Akt pathway activation, which results in the downregulation of HOTAIR expression, leading to control proliferation, invasion, and migration and inhibits apoptosis. 59In another study, it has shown the activity of calycosin suppresses some cancerous features like cell viability, migration, and invasion through downregulating Foxp3 (forkhead box P3), VEGF (vascular endothelial growth factor), and MMP-9 (matrix metalloproteinase-9) in MCF-7, T-47D cell lines for breast cancer. 60A high dose of calycosin (150 μM) tends to act against cancer by reducing its capacity to migrate and invade, while at low concentrations (20 μM) has been proven to stop the development of cancerous cells and proliferation with inducing apoptosis. 61Moreover, calycosin inhibited the proliferation, invasion, and migration of cancer cells through decreasing MMP-2, MMP-9, and CD147 levels through downregulating BATF (basic leucine zipper ATF-like transcription factor) expression and TGFβ1 (transforming growth factor 1) expression. 62In vitro study with a low concentration of calycosin has shown decreasing through induction of apoptosis of Bcl-2 (Bcell lymphoma 2) and a decrease in Bax (Bcl-2-like protein 4). 63Calycosin (100 μM) regulates the expression of ERβ, IGF-1R, PI3K/Akt, and PARP-1, inducing apoptosis and control of cell proliferation in multiple breast cancer cells. 48Studies conducted in both in vitro with Mice SKBR3 cells and MCF-7 cells, respectively, have shown upregulation of WDR7-7 (WD repeat domain 7) and downregulation of SRC (a proto-oncogene), EGFR (epidermal growth factor receptor), ERK1/2 (extracellular signal-related kinase 1/2), Akt phosphorylation that result in inhibiting proliferation and growth of cancer cells. 49Calycosin (6.25-200 μM) prevents the spread of disease and growth of MCF-7 cell cancer cells, increasing apoptosis by downregulating Bcl-2 and activating Bax and RASD1. 64Li et al. reported that in ER-positive breast cancer cells, calycosin inhibits migration and invasion of breast cancer cells with a dose of 150 μM via downregulation of Rab27B (member of the RAS oncogene family), VEGF (vascular endothelial growth factor), and βcatenin allude anti-cancer activity of calycosin. 65n conclusion, a proper dose of calycosin could inhibit the highly invasive features of breast cancer cells.Further studies might show more potentiality of calycosin against breast cancer and suggest it as a therapeutic drug among cancer patients.

| Thyroid cancer (TC)
Malignant cell formation in the tissue of the thyroid gland results in the most common endocrine tumor, that is, thyroid cancer.Calycosin has an inverse effect on thyroid cancer, for example, anti-inflammatory, antioxidant, and anti-tumor. 66Human papillary thyroid cancer cells (B-CPAP) and human normal thyroid cells (Nthy-ori 3-1) were treated with calycosin (0-100 μM), and the result demonstrated that calycosin suppresses the proliferation of cancer cells (B-CPAP). 66The general pharmacological effects on human papillary thyroid cancer cells that are related to the activation of Sestrin2 (SESN2), upregulated p-AMPK (phosphorylated AMPK), and inhibits p-mTOR (phosphorylated mTOR) and p-p70S6Kinase (ribosomal protein S6 kinase). 66verall, calycosin induced anti-tumor, antiinflammatory, and antioxidant function and decreased cancer cell proliferation while regulating many pathways in thyroid cancer therapy.

| Colorectal cancer (CRC)
A recent study on cancer data reveals the projected number of new cases of colorectal cancer in both males and females is increasing sequentially.Researchers are working to develop phytochemicals and plant derivatives anti-cancer agents to treat colorectal cancer. 67alycosin has been proven via a number of studies to slow the progression of colorectal cancer cell.Lin Zu demonstrated that calycosin could suppress the growth of xenograft mouse tumors by inhibiting the PI3K/Akt signaling pathway and increasing ER-β using siRNA and PTEN. 68Another study led by Zhao X. showed that ERβ, a mediator of the stimulation of ER-alpha, IGF-1R, p-Akt, and miR-95 expression, can suppress the proliferation of CRC cells caused by calycosin (0-80 μM) in both in vitro and in vivo model. 69Chen J. et al. declared their finding calycosin (0-100 μM) reduced cell proliferation, induced apoptosis, and upregulated of PTEN due to an elevation in ER-β as evaluated by MMT assay, flow cytometry, transwell invasion test, and mRNA levels by rt-PCR. 70Ht29 cell lines were treated with calycosin for 48 h, where calycosin upregulated caspase-3, BAX, and SIRT1, leading to activate apoptosis and autophagy. 50n another recent study, about calycosin (100 μM) work on IGF-1 stimulates growth and metastasis in CRC cell lines, where it upregulates BAX/Bcl-2, caspase 3, 9, βcatenin, BATF2 via STAT3 was with suppression of FOXM1 through STAT1 & NF-κβ pathways. 71n summary, calycosin can provide anti-colorectal cancer activities by inducing apoptosis and its powerful mechanism.Furthermore, this phytoestrogen regulates cancer-related growth factors and receptors, suppressing some signal transduction pathways and cell proliferation.

| Cervical cancer (CC)
Cervical cancer is a type of cancer that only affects women.The United States reported 14,100 new cases and 4280 fatalities in 2022. 72The dietary compounds or natural products such as quercetin (Q), sulforaphane (SFN), genistein (GEN), and epigallocatechin-3-gallate (EGCG) have an effect on cervical cell lines like NO production, expression of redox and NO pathway-associated enzymes, and apoptosis were examined. 73The MTT assay showed that calycosin caused a decrease in the viability of SiHa and CaSki cells in a dose-dependent manner (0-50 μM, 48 h) through decreasing miR-375 levels. 74Calycosin derivatives, including calycosin-7-O-D-glucoside CG (2.5-100 g/mL), dramatically decreased the expression of Bcl-2/Bax, leading to an increased apoptosis process in HeLa cells. 75Consequently, calycosin can stop the spread of cervical cancer by inducing apoptosis through several molecular mechanisms.

| Colon cancer
Colon cancer is a type of cancer that occurs in the large intestine and is caused by the abnormal growth of cells in the colon.The researchers treated colon cancer HCT-116 cells with calycosin and unraveled the effects on the proliferation of cells and apoptosis via regulation of ERβ-mediated miR-17 and PTEN expression in a dose-dependent manner. 50n summary, calycosin effectively treats colon cancer by controlling signaling pathways and suppressing cell proliferation.It also facilitated apoptosis-related cell death. 6.2.6 | Liver/hepatocellular cancer (L/HCC) Liver cancer, also known as hepatocellular carcinoma, is a kind of cancer that develops in the liver cells.Calycosin has gained attention for its anti-cancer properties relating to liver cancer.Calycosin promotes apoptosis by reducing the expression of the anti-apoptotic protein Bcl-2 and raising the expression of pro-apoptotic proteins Bax, caspase-3, and PARP and stopping the cell cycle in the G0/G1 phase through signaling pathways like MAPK, STAT3, NF-κB, and AKT at hepatocellular carcinoma (HCC) cell lines. 76Dongqing Zhang's et al. reported that calycosin appears to block cell division and decrease the length of the G0/G1 stage, potentially by downregulating the expression of TFDP1 and SKP2 genes in vitro models. 77In addition, calycosin may increase the expression of specific proteins, including transgelin 2, pyridoxine 5-phosphate oxidase, stress-induced phosphoprotein 1, peroxiredoxin 1, and biliverdin reductase B, that are involved in regulating cell proliferation and apoptosis. 77n conclusion, the available evidence suggests considering calycosin as a potential therapy for liver cancer, with multiple mechanisms of action, including anti-proliferative, pro-apoptotic, and immunomodulatory effects. 6.2.7 | Pancreatic cancer (PC) PC has a lower incidence but a greater fatality rate than other cancers, such as lung, breast, colorectal, and gastric cancer. 78alycosin inhibits cell proliferation by arresting at the S phase in the MIA PaCa-2 pancreatic cancer cell line, restoring p53 and increasing apoptosis and autophagy through the downregulation of EMT (epithelial-mesenchymal transition). 79In contrast, it was found that the expression of snail, vimentin, and CD31 was increased in the Balb/C nude mice model, suggesting that calycosin may act as an anti-cancer therapeutic agent by preventing pancreatic cancer cell proliferation. 79ccording to an in vivo study conducted on male C57BL/6N mice, calycosin (25-50 mg/kg) blocks HMGB1/NF-β signaling with decreasing production of pro-inflammatory cytokines and chemokines, notably TNF-α, IL-6, IL-1, CXCL-1, and HMGB1. 80Calycosin (25-200 μM) prevented the growth and viability of pancreatic cancer cells (PaCa-2, PANC1) by triggering p21Waf1/Cip1-induced cell cycle arrest at S phase and caspase-dependent apoptosis via caspase-3,8,9 activation, elevating Bax expression while lowering Bcl-2, consequently increasing the ratio of Bcl-2 to Bax. 81 Calycosin (100 μM) diminished cyclin A1 and the related cyclin-dependent kinase CDK2 expression while increasing Snail, MMP-2, -9, and p21 gene expression and regulated Raf/MEK/ERK pathway in the pancreatic cell line MIA PaCa-2. 81Calycosin regulated MUC1, Atg5 protein expression snail protein, G1 phase arrest, and TGF-β in MIA-PaCa2 cell line at 50-100 μM, leading to cause apoptosis and autophagy, while at 30 mg/kg prevent tumor growth in C57/BL6 mice. 82Additionally, in Balb/C mice, calycosin treatment (25-50 mg/kg) hindered NF-Bҡ/ p65 expression, phosphorylation of an IκBα and p38 MAPK, and decreased the level of tumor necrosis factor TNF-α, IL-6, and IL-1.Besides this, it suppressed MPO activity, improved SOD (superoxide dismutase) activity, and caused apoptosis in mice with acute pancreatitis. 83argely, calycosin can limit cell growth and induce apoptosis via activating the caspase pathway, both of which have anti-pancreatic cancer properties.Additionally, this phytoestrogen controls growth factors and receptors linked to cancer while also inhibiting some signal transduction pathways. 6.2.8 | Glioblastoma multiform (GMB) The major and lethal malignant brain tumor in adults is glioblastoma (GBM), consisting of 16% of all primary brain and central nervous system tumors.It is challenging to diagnose and treat because most drugs are not permeable to the bloodbrain barrier.However, in recent years, calycosin has been used to treat glioblastoma effectively.Calycosin (50-800 μM) in U251 and U87 cells can downregulate c-Met, MMP9, Dtk, Lyn, and PYK2 and p-AKT expression, resulting in suppression of cell proliferation, invasion, and induce cell death. 27In further studies, calycosin inhibits glioblastoma cell proliferation and invasivion, migratory properties by decreasing downstream inflammatory cytokines, including NLRP3, NF-B, and IL-1 in glioma cell lines U251, U87, and BALB/c mice. 84Calycosin (0-800 mM) downregulates the TGF-β leading to the inactivation of EMT, MMP-2, and MMP-9 and activating N-cadherin, Snail, and Vimentin in U87 and U251 cell lines. 85In another study, calycosin reduced cell viability in U87 cells by exerting antioxidant effect through the Wnt/GSK3−/catenin signaling pathway, regulating SOD, CAT, and GSH levels, induced apoptosis by activating the mitochondria-associated caspase pathway and regulating pGSK-3βser9, catenin, and c-Myc at 1-100 μg/ mL. 86In treating C6 malignant glioma, the intricate formononetin and calycosin (FMN/CAL) components showed a chemosensitization impact on temozolomide. 87Co-administration of the three drugs may reduce cell growth and increase apoptosis by increasing the expression of these apoptotic proteins: Bax, Cleaved Caspase-3, Cleaved Caspase-9, and downregulating anti-apoptotic protein Bcl-2 at dose-dependent manner. 88n short, naturally occurring calycosin inhibits the growth, multiplication, and invasion of glioblastoma cells by activating certain proteins linked to apoptosis.Co-administration of medications with others promotes anti-cancer activity. 6.2.9 | Gastric cancer (GC) According to cancer statistics from the USA for 2022, this cancer had a mortality rate of 11,090 and a new case rate of 26,380. 72The use of Chinese compound medicines like QLD contains a significant amount of calycosin and may be able to prevent the migration and invasion of gastric cancer cell lines. 89Zhang Y. et al. investigated the effects of calycosin (47 μM) on increased p27 and p21 expression levels and cell cycle at G0/G1 with lowering cyclin D1, cyclin E, and CDK2, CDK4, and CDK6 levels, in gastric cancer. 90Li D. et al. reported that calycosin downregulated the elevated NF-ҡB, p-NF-B, DARPP-32, and STAT3 in vitro model, while it (40-80 mg/kg) reduced integrin 1 in vivo study. 91Zhou L. et al. found that cisplatin/5-FU/ADM paired with calycosin (10 g/ mL) suppressed Akt phosphorylation in cell lines SGC7901, BGC823, and NCIN87 from the human stomach. 28hus, calycosin has the possibility as a potential therapeutic agent for gastric cancer through preventing migration and invasion of cancer cell by downregulating growth factor and increasing tumor suppressing proteins expression and cell cycle arrest. 6.2.10 | Nasopharyngeal cancer (NPC) Globally, one of the most prevalent malignant tumors is nasopharyngeal carcinoma (NPC), which is caused due to genetic factors, EB virus infection, external conditions, and other factors. 92,93The pharmaceutical drugs to manage NPC are inadequate, especially for patients with advanced condition. 69,94A recent investigation in vitro study demonstrates that calycosin effectively inhibits the proliferation of NPC cells by altering the expression of the lncRNA Ewing sarcoma-associated transcript 1 in Ewing sarcoma cells. 95Treating NPC cells with calycosin (20-50 μM) showed a decreased expression of ESWAT1, p-TAK1, p-IкB, and p-c-Jun and suppression of NPC cells by mediating downstream effectors TRAF6 and TAK-1. 92Calycosin increases tp53 and caspase 8 levels and decreases MAPK14 expression in a dose-dependent way resulting in the apoptosis of NPC cells and halting proliferation and the growth of tumor cells. 94.2.11 | Ovarian cancer (OC) Ovarian cancer is a kind of cancer that begins in the ovaries.Patients with ovarian cancer typically undergo surgery as their first line of treatment, which usually entails a combination of hysterectomy, bilateral salpingooophorectomy, and removal of the omentum, and platinum compounds. 96,97Therefore, creating a cuttingedge treatment for ovarian cancer is urgently needed. 98t has been determined that calycosin contains powerful anti-cancer pharmacological properties and possesses anti-cancer function against ovarian cancer via numerous mechanisms.Y. Zhou and his colleagues found that calycosin (100 μM) inhibited SK-0V-3 cancer cell growth, proliferation and induced apoptosis via upregulating production of cleaved caspase-3, cleaved caspase-9, Bax/ Bcl-2 ratio and downregulating expression of Bcl-2, procaspase-3, pro-caspase-9 signaling. 98Calycosin (50 μM) therapy increased cancer cell apoptosis in the SK-0V-3 cell line via upregulating caspase-3 expression and combining Cisplatin and calycosin induced apoptosis via upregulating expression of several signaling molecules such as P53, caspase-3, caspase-9, Bax, and Bcl-2. 99n conclusion, calycosin may operate as an antigrowth and apoptosis-inducing agent against ovarian cancer SKOV3 cells by activating caspases and Bcl-2 family proteins and the p53 pathway making it a prospective therapeutic agent for the treatment of ovarian cancer. 6.2.12 | Osteosarcoma (OS) Osteosarcoma (OS) is a cancerous neoplasm of the bone that primarily affects teenagers, children, and young adults.Like other cancer, calycosin possesses anticancer activities against osteosarcoma in various ways.
Calycosin induces apoptosis by upregulating lactate dehydrogenase (LDH) content, caspase-3, and downregulating miR-223, PCNA, Bcl-2, PARP, NF-κB, p65, and IκBα signaling molecules in 143B cell line, where in vivo study of tumor-xenograft nude mice, it induces apoptosis and reduces tumor cell mass via downregulating signaling components such as miR-223, NF-κB, p65, IκBα, Bcl-2, and PARP; it mainly suppresses the neoplastic miR-223-IκBα pathway. 100Qiu et al. conducted a study on BALB/c nude mice at various doges (30-120 mg/kg) and found that calycosin reduced tumor weight via downregulating MPP2, IκBα and ECT2, IL-6 signaling components, simultaneously activate apoptosis, suppressed cell proliferation, metastasis, and cell growth at various doges (60-180 μmol/L) in 143B human cell line. 101Tan et al. found that calycosin induced apoptosis and reduced cell proliferation and growth in the U2OS human cell line by upregulated the expression of TP 53 and CASP3 and downregulated the expression of XIAP. 57n conclusion, the current results show that calycosin has pharmacological effects against osteosarcoma (OS) by both in vivo and in vitro inhibiting the neoplastic IκBa/ ECT2 pathway and suppressing the neoplastic miR-223-IκBα pathway, both of which are connected to triggering death in tumor cells. 6.2.13 | Lung cancer (LC) Although lung cancer is life-threatening cancer, effective treatments are not available.Some studies depicted the impact of calycosin against lung cancer as it possesses antitumor, neuroprotective, and anti-inflammatory properties. 30UAD cells (A549 and H1299) were treated with different concentrations of calycosin, which could cause an increased level of cir_0001946, miR-21 expression, and decreased HIF-1α. 102Moreover, calycosin treatment at A549 cells showed a significant anti-tumor activity through the underline mechanism of repressing MMP-2 and MMP-9 expression levels and inhibiting cell proliferation via controlling cell signaling molecules, including phosphorylation of ERK1/2 along with low levels of integrin β1 at the same cell lines. 103n conclusion, the expansion and migration of lung cancer cells were inhibited by the pharmacological effect of Cal via E-cad/TPA/MMP-2/Annexin V+/PI-cells signaling axis in a dose-dependent way.

| Leukemia
Leukemia is a type of blood cancer that is characterized by the rapid development of abnormal blood cells.Dongqing Zhang and his colleagues studied the effects of calycosin downregulate expression of Cyclin D1 to arrest cell cycle at G0/G1 phase leading to inhibit cell proliferation at K562 cell lines. 104 6.205 Calycosin inhibits melanogenesis by reducing the activity of the PKA/CREB and p38 MAPK signaling pathways. Itmainly inhibits the tyrosinase activity and downregulates the expression of MITF and TRP-2 protein.106 Summary of chemotherapeutic activity of calycosin against human malignancies is presented at Table 3. Graphical presentation of anti-cancer mechanism mediated by calycosin through different mechanism including apoptosis, angiogenesis and metastasis, cell signaling and proliferation and miRNA regulation is presented at Figures 2-4.

| Clinical trial of calycosin in cancer treatment
Calycosin has been proven to own powerful anti-cancer properties in silico, in vitro, and in vivo.However, report from a clinical trial of calycosin with cancer is insufficient.A clinical case report conducted by Qiu et al. with osteosarcoma patients reported enhanced levels of neoplastic miR-223, and elevated expressions of NF-κBp65, IκBα proteins in tumor cells compared to non-OS control. 100owever, 143B cells cell cultures from this patient treated with calycosin downregulate miR-223 level, Bcl-2, PARP, NF-κB, p65, and IκBα, while lactic dehydrogenase (LD) content, and caspase-3 positive cells were elevated. 100oreover, a controlled randomized trial study conducted by Wang et al. summarized that calycosin accelerated the apoptosis of the LX-2 cell line but not in cancer cells. 107

| POTENTIAL SYNERGY OF CALYCOSIN WITH OTHER AGENTS IN CANCER TREATMENT
It has already been established that using natural items to treat cancer has excellent therapeutic potential.These phytochemicals work remarkably well against cancer when combined with standard drugs or other phytochemicals, reducing the adverse effects of many other medications.Regarding glioma treatment, calycosin potentially works in conjunction with temozolomide to decrease tumor density by inhibiting cell migration, inducing cell death, and upregulating apoptotic proteins' expression, including Bax, cleaved caspase-3, cleaved caspase-9 and decreased the expression of migratory proteins in the C6 xenograft mouse model. 88Calycosin has a good combination with cisplatin, which inhibits the proliferation of laryngeal squamous cell carcinoma and enhances the chemosensitivity of cancer cells to cisplatin. 108Moreover, a combination of this compound is effective against gastric cancer cells by preventing protein kinase B phosphorylation (Akt). 28Calycosin from Chinese medicine formula (CCMF) exerts synergism via way of the activation of PP2A and inhibition of UPS, activating the mitochondrial apoptosis pathway against multiple cancers, including hepatocellular carcinoma and colorectal cancer. 109However, the toxicity and side effects brought on by employing CDDP alone are diminished since its derivative calycosin-7-O-Dglucoside considerably boosts the CDDP-induced death of the SK-OV-3 cells through the p53 pathway at the cellular level. 99Calycosin is also effective with a combination of others phytochemicals.For example, a good combination of formononetin/calycosin can enhance the sensitivity of malignant glioma to TMZ by downregulating NOS2dependent cell survival in C6 cells, which provides a basis for the application of this combination in the adjuvant treatment of glioma. 87Additionally, Zhang et al. described that a combination of these compounds promoted tumor cells apoptosis through the expression of Bax, cleaved caspase-3, and cleaved caspase-9, decreased the expression of Bcl-2 and inhibiting migration through the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in C6 cell. 110

IN ALLEVIATING MULTIDRUG RESISTANCE IN NUMEROUS CANCER TYPES
Multidrug resistance (MDR) is a significant obstacle in the fight against cancer.Sometimes anti-cancer drugs have some level of resistance, which can appear through a variety of mechanisms, such as reduced drug uptake, increased drug efflux, activation of detoxification systems, activation of DNA repair processes and many more. 111any proteins, including P glycoprotein (P-GP), MRP 1, MRP 1-9, BCRP, and changes in beta-tubulin, are linked to the occurrence of drug resistance. 112 increase the suppression of cisplatin to gastric cell lines by preventing protein kinase B from being phosphorylated (Akt).Hence, it is possible to achieve a better therapeutic effect in a lower concentration when cisplatin/5-FU/ADM is coupled with calycosin. 28Epithelial-to-mesenchymal transition (EMT) is an important target to overcome drug resistance.Fortunately, calycosin suppresses TGFβinduced epithelial-to-mesenchymal transition leading to upregulated expression of BATF2 via the STAT3 pathway by suppressing TGF-β which controls PCNA expression through the phosphoinositide 3-kinase pathway in colorectal cancer cells. 51IFN-γ's backbone folded into a more packed structure with increased -helix content and a higher melting temperature (Tm), which has an antiproliferative impact as a result of the interaction between calycosin and IFN-γ against HepG2 cells, which are hepatocellular carcinoma, by activating caspase-9/3. 113The combination of calycosin and formononetin decreased the expression of NOS2 in tumor tissues and cells and decreased TNF-α production in the tumor area. 87By stimulating ROS-mediated MAPK, STAT3, and NF-B signaling pathways, calycosin inhibited the expression of TGF-1, SMAD2/3, SLUG, and vimentin and resisted hepatocellular cancer. 76Zhu Zhang et al. showed that calycosin caused p21Waf1/Cip1-induced cell cycle arrest and caspasedependent apoptosis while stimulating the migration of MIA PaCa-2 cells by inducing the epithelial-mesenchymal transition (EMT) and activating matrix metalloproteinase in pancreatic cancer. 81Furthermore, in glioblastoma cell lines, calycosin drastically improve the expressions of mesenchymal-associated genes/activators, such as Ncadherin, Snail, and vimentin, and modulated TGF-β. 85alycosin may have inhibitory effects on ER breast cancer since it at least partially reduces the growth of breast cancer cells through WDR7-7-GPR30 signaling. 49Moreover, Songya et al. summarized that creating temozolomide as a treatment option for brain tumors with a combination of formonectin/calycosin could improve the sensitivity of C6 malignant glioma to TMZ by preventing NOS2-dependent cell survival. 87In their in silico study, Huang et al. showed that calycosin alters the expression of some multi drugs resistance proteins, including ATP-binding cassette subfamily G member 2 (ABCG2) and breast cancer type 1 susceptibility protein (BRCA1) in colorectal cancer. 54verview of synergistic activity and reversing resistance mechanism of calycosin with other phytochemicals and conventional chemotherapeutic agents is shortlisted at Table 4.The graphical presentation of synergistic activity and reversing resistance mechanism of calycosin is summarized at Figure 5.

STRATEGIES OF CALYCOSIN WITH AIMING BETTER BIOAVAILABILITY
Despite having the well-known therapeutic application of calycosin, it has poor solubility and bioavailability, which can regret its biological activities. 114However, this challenge can be solved via nanotechnology because combining phytochemicals with nanocarriers makes them more soluble. 115Nanocarriers that have received clinical approval are used to treat and diagnose numerous diseases, including cancer. 116diabetic nephrology, 114 and ischemic heart disease. 117Calycosin performed in vitro and in vivo studies with nanostructured lipid carriers against cancer.The surfactants Tween 80, Span 60, PEG 400, and sucrose stearate were administered to the MDA-MB-231 cell line before loading calycosin.The drug-loaded NLCs' average particle size, PDI, zeta potential, spherical shape, 89% drug encapsulation, and 6.5% drug loading capacity were all within the range of 100 nm. 116Although calycosinnanocarriers related to cancer have limited information, studies with diseases have been done.One of the reasons individuals with acute myocardial infarction experience unsuccessful therapy is insufficient medication.Rats were used in a recent study employing phytochemicals in nano-systems, and the results indicated the best infarct size reduction and maximum heart accumulation.Mitochondrion-targeted tetrapeptide (MTP) and cyclic arginyl-glycyl-aspartic acid (RGD) peptide co-modified nano-system were employed as treatments for co-loaded calycosin (CAL) and tanshinone (TAN). 118For the treatment of diabetic nephropathy, Hung et al. conducted an in vivo and in vitro experiment using free and calycosinloaded nanoliposomes.Rats were given an oral dosage of 30 mg/kg; pharmacokinetics activity was improved. 114ecent investigations have demonstrated that calycosin can be utilized as a medication, and clinical trials may be carried out using them as the foundation.

TOXICITY OF CALYCOSIN
Although calycosin offers many medicinal benefits, the human body may experience only minor adverse effects or harmful responses.Calycosin favors cancer cells when it is present in modest concentrations, including promoting the growth of cancer cells, reducing apoptosis, and downregulating ERK 1 and 2. 61 In vivo study also showed that calycosin stimulates a dramatic increase in urinary weight. 29According to previous research, calycosin promotes angiogenesis in zebrafish larvae and human umbilical vein endothelial cells (HUVEC) both in vitro and in vivo.The zebrafish model also demonstrated that calycosin regulates the ErbB signaling pathway, VEGF (vascular endothelial growth factor), and FGF. 119No additional in vivo tests revealed calycosin harm, confirming its efficacy as a treatment. 21To determine whether or not calycosin can be harmful by blocking digestive enzymes, its medicinal impact should be thoroughly assessed. 21

| PHARM ACO KIN ETICS OF CALYCOSIN AND FUTURE PERSPECTIVE IN DRUG DEVELOPMENT
In silico approaches were used to conduct pharmacokinetics or ADME/Tox prediction through computational tools such as Schrodinger's QuickPro modules, online accessible server admetSAR, and SwissADME. 4,120It was observed that molecular weight, QPlogPo/w, HBD, and HBA of calycosin were 284.26 g/mol, 1.5, 2, and 5, respectively, indicating good drug-likeness properties of the compounds.Molecular weight (MW), volume, density, nHA, nHD, nRot, nRing, MaxRing, nHet, fChar, nRig, flexibility, stereo centers, TPSA, logS, logP, and logD were among the physicochemical characteristics radar of calycosin that were computed using the ADMETlab 2.0 software and were shown in Figure 6.In deciding whether or not to move a substance to the clinical stage, evaluating drug absorption, distribution, metabolism, elimination, and toxicity (ADMET) forecast may help minimize the risk, time, and expense involved.Table 5 summarizes the pharmacokinetic profile of calycosin, predicted using in silico tools like Schrodinger's QuickPro modules and the online server admetSAR, which provides reliable data about absorption, distribution, metabolism, excretion, and toxicity.
Drugs must possess the ability to absorb to reach the bloodstream and be used in daily life.Our predicted result showed that calycosin might be highly absorbed orally and can be permeable by skin, intestine, and kidney cells.Distribution is the movement of a substance within the body and is influenced by several variables, including the permeability of the blood-brain barrier, the permeability of the central nervous system, the binding of the drug to plasma proteins, and the overall volume of distribution.Drugs that attach to plasma proteins (such as human serum albumin, lipoprotein, glycoprotein, and globulins) significantly decrease the amount of the substance in the bloodstream.The fact that our intended chemicals fall within the suggested range suggests that they are likely to move quickly in circulation and reach the target location.The quantity of body fluid needed to disperse in blood plasma is known as the volume of diffusion.Fortunately, calycosin has the probability of distributing uniformly in tissue and blood plasma.In addition, the blood-brain barrier (QPlogBB) and central nervous system are essential for drugs targeting the brain disorder.Unfortunately, calycosin has a poor potential to cross the brain-blood barrier and central nervous system.The CYP450 enzyme detoxifies more than 80% of medications during hepatic first-pass metabolism, and blocking this enzyme increases drug potency and several adverse effects.The predicted metabolic result reported that calycosin is a substrate for most CYP450 subunits, including CYP1A2, CYP2C9, and CYP2D6.Calycosin's in silico toxicity profile has been predicted based on hepatotoxicity, cardiotoxicity, skin sensitization, kidney toxicity, eye irritation, and AMES toxicity, where calycosin has shown toxicity against ll except AMES toxicity.We used total clearance (CLtot) and T 12 to estimate the clearance profile based on the compounds' kidney clearance characteristics.As anticipated, the findings showed that calycosin can be easily eliminated from the body once it has served its intended medicinal purpose.
Therefore, pharmacokinetics profile of calycosin suggests good lead and efficiency with fewer toxicity risks.So, calycosin can be a good starting source of drug design, such as docking and pharmacophore-based virtual screening in cancer treatment and drug discovery communities.

LIMITATIONS OF THIS STUDY
Based on the evidence presented in this current review, calycosin may be a prospective molecular lead for developing anti-cancer drugs.As far as we know, this is the first review article with calycosin against numerous cancers.This compound mediated anti-cancer activities against multiple cancer due to the specificity of some receptors, including estrogen receptor, IGF-1R, and EGFR.The detail mechanism of anti-cancer activity from in silico, in vitro, preclinical, and clinical studies suggested that calycosin mediated its anti-cancer efficacy through targeting apoptosis protein, anti-apoptotic proteins, among others (Bcl-2) and apoptotic proteins (Bax, Bak, caspase), arresting cell cycle as well as cell proliferation, including inhibiting expression and activity of cyclins (A1, D1, E) and CDKs (2, 4, 6), or increasing the expression of CDKs inhibitors (p21, p27, p53), metastasis and angiogenesis (MMP-2& 9, Wnt/-catenin, PARP), regulating inflammation (TNF-α, NF-κB, IκB kinase, IL-1β, phospho-Akt, phosphor-p65), targeting cell signaling (NF-κB, PI3K/Akt, MAPK/ERK, p-mTO) in numerous cancer cell lines (Figure 7).Also, it has been shown that calycosin synergistically increases the anti-cancer activities of other natural products and some conventional drugs after conjugation with them and alleviates the resistance profile of anti-cancer drugs.Information on calycosin about nanotechnological approaches in cancer treatment is limited, so further studies with calycosin nanotechnology are needed to improve target cancer tissues.Furthermore, having good pharmacokinetics with lesser toxicities suggests that this natural compound may become a promising drug discovery candidate after using other techniques like network pharmacology and molecular docking against many diseases, especially cancer.It's worth mentioning that while these potential mechanisms of anti-cancer activity are supported by preclinical studies conducted in cell lines and animal models, more research is needed to establish the effectiveness of calycosin in human cancer therapy.Clinical trials are essential to determine its safety, optimal dosage, and actual benefits in treating various types of cancers.

F I G U R E 2 AF I G U R E 3
general mechanism for calycosin-mediated apoptosis in human malignancies.Calycosin mediates entry through its receptor and activate Caspase 8, inactivate PARP.Furthermore, it increases ROS production and releases Cyt-c and apaf-1.All of these combined effects induce death of cancer cell.The impact of calycosin on angiogenesis and metastasis in cancer cells.Figure outlines the progression of angiogenesis and metastasis by sequential mechanism.Calycosin interfere at angiogenesis by suppressing angiogenetic factor, including VEGF, MMPs, TGF-β, VEGF, and TNF-α, cell adhesion molecules such as E-cadherin-cadherin and Vimentin and invasion to blood vessel.

F I G U R E 4
Calycosin modulate miroRNA (miRNA) to fight cancer cells.After interring to the nucleus, it directly alter the transcription and alter miRNA production.This sequence lead to translate some anti-cancer proteins like apoptosis, cell proliferation, cell viability, hypoxia, and signaling molecules.TA B L E 4Overview of synergistic activity of calycosin with other phytochemicals and conventional chemotherapeutic agents.

F I G U R E 5
Diagrammatic representation of overcoming multidrug resistance by calycosin via targeting signaling mechanism.

F I G U R E 6
Assessment of physicochemical properties of calycosin.
41ony root contains calycosin and has been researched for its anti-inflammatory and potential anti-cancer properties.It may inhibit the growth of certain cancer cells.41

Cancer types Receptor interaction Molecular basis Experimental model Ref
Molecular interaction of calycosin with numerous receptors leading mediate anti-cancer activities.
Huang et al. showed T A B L E 2

Descriptors Predicted remarks Comments Predicted value Recommended rage
In silico drug-likeness and pharmacokinetics properties profile of calycosin.