NPEPPS Is a Druggable Driver of Platinum Resistance.

There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder cancer, platinum-based chemotherapy leads to better outcomes in a subset of patients when used in the neoadjuvant setting or in combination with immunotherapy for advanced disease. Despite such promising results, extending the benefits of platinum drugs to a greater number of patients is highly desirable. Using the multiomic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified puromycin-sensitive aminopeptidase (NPEPPS) as a driver of cisplatin resistance. NPEPPS depletion sensitized resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. NPEPPS affected treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDO) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin, which was concordant with clinical response. In the PDOs, depletion or pharmacologic inhibition of NPEPPS increased cisplatin sensitivity, while NPEPPS overexpression conferred resistance. Our data present NPEPPS as a druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations. SIGNIFICANCE: Targeting NPEPPS, which induces cisplatin resistance by controlling intracellular drug concentrations, is a potential strategy to improve patient responses to platinum-based therapies and lower treatment-associated toxicities.

The potential therapeutic effect of melatonin in oxaliplatin combination therapy against chemoresistant colorectal cancer cells.

BACKGROUND: Oxaliplatin is one of the main therapeutics in colorectal cancer (CRC) chemotherapy. However, in light of multidrug resistance (MDR) phenotype development, the efficacy of oxaliplatin has decreased. This study aimed to assess the potential therapeutic effect of melatonin in oxaliplatin combination therapy for drug-resistant colorectal cancer cells. METHODS AND RESULTS: Initially, the oxaliplatin-resistant cell line was created of LS174T (LS174T/DR) by using the oxaliplatin IC50 concentration and resting cycles. MTT assays and flow cytometry were applied for assessing cell viability and apoptotic cells. The mRNA expression level of Bax, Bcl2, MT1, MT2, and ABCB1 as well as protein levels of ABCB1, Bcl2, BAX were measured by the qRT-PCR and western blot techniques respectively. P-gp activity was assessed by Rho123 staining. The IC50 concentration of oxaliplatin in resistant cells was increased from 500.7 ± 0.2 nM to 7119 ± 0.1 nM. Bcl2, MT1, MT2, and ABCB1 mRNA plus protein expression levels of Bcl2 and ABCB1 were significantly reduced in resistant cells, along with a marked increase in Bax mRNA and protein levels compared to parental cells. Rho 123 staining revealed a marked reduction in P-gp activities in the combination-treated group compared to the oxaliplatin-treated group. CONCLUSIONS: The results of cytotoxicity assays, MTT, and flow cytometry revealed that the combination of melatonin and oxaliplatin exerts synergistic effects on induction of oxaliplatin's cytotoxicity in CRC. Our research suggests that combining the treatments of melatonin and oxaliplatin may be considered as a new approach to overcoming oxaliplatin resistance in CRC patients.

Vandetanib alters the tumoricidal capacity of human breast cancer stem cells via inhibiting vasculogenic capacity.

Introduction: The inhibition of vascularization into tumor stroma as well as dynamic cell growth is the center of attention. Here, we aimed to examine the role of vandetanib on angiogenesis capacity of breast cancer stem cell (CSCs). Methods: MDA-MB-231 cells were exposed to different doses of vandetanib and survival rate was monitored. Stimulatory effects of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were evaluated in vandetanib-treated MDA-MB-231 cells. In vitro tubulogenesis capacity was studied on the Matrigel surface. The synergistic effects of vandetanib on cell survival were also assessed after PI3K and/or Wnt3a inhibition. Vascular endothelial (VE)-cadherin, matrix metalloproteinase-2 (MMP-2), -9, Wnt3a, and p-Akt/Akt ratio were measured using western blotting. Results: Vandetanib reduced survival rate in a dose-dependent manner (P<0.05). Proliferative effects associated with VEGF, FGF, and EGF were blunted in these cells pre-exposed to vandetanib (P<0.05). The microcirculation pattern's triple-negative breast cancer (TNBC) was suppressed by 1, 5 µM of vandetanib (P<0.05). Hence 1, 5 µM of vandetanib potentially decreased the population of CD24- cells. 1 and 5 µM of vandetanib inhibited cell proliferation by blocking PI3K and Wnt3a pathways and decreased the p-Akt/Akt ratio, Wnta3 protein levels (P<0.05). 1 and 5 µM vandetanib combined with PI3K inhibitor diminished metastatic markers including, MMP-2, and MMP-9. The concurrent treatment (PI3K, inhibitor+ 1, 5 µM vandetanib) also considerably reduced epithelial-mesenchymal transition (EMT) markers such as VE-cadherin (P<0.05). Conclusion: Vandetanib suppressed vasculogenic mimicry (VM) networking through blunting stemness properties, coincided with suppression of VE-cadherin in CSCs.

The Impact of Covid-19 Pandemic on Genitourinary Cancers Stage and Grade.

INTRODUCTION: Our study aims to evaluate the impact of the COVID-19 pandemic on the number of uro-oncological surgeries (cystectomy, nephrectomy, prostatectomy, orchiectomy, and transurethral resection of bladder tumor (TURBT)) and pathological staging and grading. MATERIALS AND METHODS: The present study is a retrospective study on patients with genitourinary cancers treated from 2018 to 2021 in a referral tertiary center. The data were obtained from the hospital records with lengths of 22 and 23 months, labeled hereafter as non-COVID and COVID pandemic, respectively (2018/3/21-2020/1/20 and 2020/1/21-2021/12/21). The total number of registered patients, gender, age, stage, and grade were compared in the targeted periods. Moreover, all the pathologic slides were reviewed by an expert uropathologist before enrolling in the study. The continuous and discrete variables are reported as mean (standard deviation (SD)) and number (percent) and the χ2 test for the comparison of the discrete variables' distribution. RESULTS: In this study total number of 2077 patients were enrolled. The number of procedures performed decreased during the Covid pandemic. The tumors' distribution stage and grade and patients' baseline characteristics were not significantly different in non-COVID and COVID pandemic periods for Radical Nephrectomy, Radical Cystectomy, Radical Prostatectomy, and orchiectomy. For TURBT only, the tumor stage was significantly different (P-value<.001) from the higher stages in the COVID pandemic period. CONCLUSION: Among urinary tract cancers, staging of bladder cancer and TURBT are mainly impacted by the COVID-19 pandemic with higher stages compared to the non-COVID period. We evaluate the impact of the COVID-19 pandemic on the number of uro-oncological surgeries based on pathological staging and grading. Total number of 2077 patients were enrolled. Among urinary tract cancers, staging of bladder cancer and TURBT are mainly impacted by the COVID-19 pandemic with higher stages compared to the non-COVID period.

Exercise and colorectal cancer: prevention and molecular mechanisms.

Exercise and physical activity have been shown to be strongly associated with a decreased incidence rate of various chronic diseases especially numerous human malignancies. A huge number of clinical trials and meta-analysis have demonstrated that exercise is significantly effective in lowering the risk of colorectal cancer. In addition, it is suggested as an effective therapeutic modality against this cancer type. Therefore, in this review, we will review comprehensibly the effects of exercise in preventing, treating, and alleviating the adverse effects of conventional therapeutic options in colorectal cancer. Moreover, the possible mechanisms underlying the positive effects of exercise and physical activity in colorectal cancer, including regulation of inflammation, apoptosis, growth factor axis, immunity, epigenetic, etc. will be also discussed.

Isolation of cfDNA from spent culture media and its association with implantation rate and maternal immunomodulation.

OBJECTIVES: This investigation aims to evaluate the association between the concentration of cell-free DNA (cfDNA) in the spent culture medium (SCM) with implantation rate and the maternal immune system in the invitro fertilization (IVF). In this study, 30 embryos were cultured and scored according to Gardner's criteria. SCM was gathered on day five from every embryo to analyze the quantity of cfDNA. The real-time PCR technique evaluated the expression level of transcription factors, including Foxp3, RORγt, GATA3, and T-bet. The percentage of Th1, Th2, Th17, Treg, NK cells, and NK cells cytotoxicity was evaluated by flow cytometry. RESULTS: The concentration of cfDNA in the ß-HCG (-), ß-HCG ( +), and ongoing pregnancy groups were 20.70 ± 9.224 ng/µL, 27.97 ± 7.990 ng/µL, and 28.91 ± 8.566 ng/µL, respectively. The ratio of Th1/Th2 and Th17/Treg reduced significantly in pregnant women, as well as the level of NK cells and NK cytotoxicity cells fell dramatically in the ongoing pregnancy group. The expression level of RORγt and T-bet declined while the expression level of Foxp3 and GATA3 increased considerably in pregnant mothers. Our investigation revealed that the concentration level of cfDNA in SCM could not be associated with implantation rate, prediction of ongoing pregnancy, and maternal immune system.

An evaluation on potential anti-oxidant and anti-inflammatory effects of Crocin.

Crocin, an active ingredient derived from saffron, is one of the herbal components that has recently been considered by researchers. Crocin has been shown to have many anti-inflammatory and antioxidant properties, and therefore can be used to treat various diseases. It has been shown that Crocin has a positive effect on the prevention and treatment of cardiovascular disease, cancer, diabetes, and kidney disease. In addition, the role of this substance in COVID-19 pandemic has been identified. In this review article, we tried to have a comprehensive review of the antioxidant and anti-inflammatory effects of Crocin in different diseases and different tissues. In conclusion, Crocin may be helpful in pathological conditions that are associated with inflammation and oxidative stress.

Application of cell free DNA in ART.

Various biopsy and sampling methods are used for preimplantation genetic diagnosis (PGD) of embryo. This method benefits blastomer/trophectoderm biopsy to improve the clinical outcome of in vitro fertilization (IVF). However, all of these procedures are invasive and have adverse effects on embryo development. Additionally, these procedures require expensive equipment and well-experienced technicians. Regarding these limitations, designing non-invasive methods is necessary. One of the recently proposed non-invasive and applicable methods is cell free DNA (cfDNA) molecule evaluation that have opened up exciting opportunities in the molecular diagnosis of embryo and fetus chromosomal aneuploidy. cfDNA is present in body fluids; especially blood, follicular fluid, amniotic fluid, spent embryo culture medium (SCM) and blastocoel fluid. Overall, this review highlights the cfDNA biomarker might constitute a supplemental tool for improving IVF and pregnancy outcomes, female infertility management. However, the successful application of cfDNA demands an understanding of its biological properties, kinetics, time of collection, high sensitivity and specificity cfDNA detection methods, and their limitation and challenges in the clinical settings. In this review we also describe ethical aspects of cfDNA testing.

Effect of Apatinib plus melatonin on vasculogenic mimicry formation by cancer stem cells from breast cancer cell line.

BACKGROUND AND AIM: Vasculogenic mimicry (VM) is one of the most important causes of breast cancer metastasis and resistance against drugs. The cancer stem cells (CSCs) are known as essential factors for VM formation. In this study, the effects of melatonin, Apatinib, and a combination of Apatinib/melatonin on VM formation were investigated by breast CSCs from breast cancer cell line. MATERIALS AND METHODS: The percentage of CSCs was determined in two breast cancer cell lines (MCF-7 and MDA-MB-231) by flow cytometry. The effects of Apatinib, melatonin, and a combination of Apatinib/melatonin were evaluated on proliferation and viability, migration and invasion, apoptosis, and VM formation in MDA-MB-231 cells. Moreover, expression levels of the involved proteins in cancer cell proliferation and viability, CSCs, migration and invasion, and VM formation were evaluated by real-time polymerase chain reaction (RT-PCR) and western blotting methods. RESULTS: Results of the present study showed that melatonin and Apatinib reduced survival rate of CSCs in a dose- and time-dependent manner. Apatinib, melatonin, and a combination of Apatinib/melatonin inhibited proliferation of breast CSCs (P ≤ 0.001). Formation of VM was decreased in the MDA-MB-231 cancer cell line treated with Apatinib and combination of Apatinib/melatonin. Apatinib and combination of Apatinib/melatonin reduced invasion of breast CSCs (P ≤ 0.0001). Expression of vascular endothelial VE-cadherin, ephrinA2 receptor (EPHA2), p-PI3K/phosphoinositide-3 kinase (PI3K) and phospho-AKT (p-AKT)/AKT ratios was decreased under the influence of Apatinib and a combination of Apatinib/melatonin (P ≤ 0.01). CONCLUSION: Apatinib or a combination of Apatinib/melatonin may be used to manage patients with breast cancer. However, further studies are needed to identify anti-cancer mechanisms of melatonin and Apatinib for better management of the patients with breast cancer.

Combination of Estradiol with Leukemia Inhibitory Factor Stimulates Granulosa Cells Differentiation into Oocyte-Like Cells.

Purpose: Previous studies have documented that cumulus granulosa cells (GCs) can trans-differentiation into different non-ovarian cells, showing their multipotentiality to repopulate the injured cells in ovarian tissue. The current experiment is aimed to assess the differentiation capacity of human cumulus GCs toward the oocyte-like phenotype in vitro. Methods: GCs were isolated from healthy female volunteers subjected to in vitro fertilization or intra-cytoplasmic sperm injection (IVF-ICSI). The effect of different media supplemented with leukemia inhibitory factors (LIFs), 5 ng/mL estradiol, and 0.005 IU/mL follicle-stimulating hormone (FSH) were investigated to the differentiation of GCs toward oocyte-like phenotype via monitoring the expression of Oct3/4 and GATA-4 using flow cytometry analysis. The expression of genes such as FIGLA, NOBOX, and SYCP3 was measured by real-time polymerase chain reaction (PCR) assay. We also assess morphological adaptation by using bright-field microscopic imaging. Results: Exposure of GCs to LIFs increased the number of cells expressing stemness factor Oct3/4 coincided with the suppression of GATA-4 after 7 days (P < 0.05). We found that the transcript level of all genes FIGLA, Nobox, and SYCP-3 decreased in cells after treatment with a FSH (P < 0.05). According to our data, the incubation of GCs with estradiol increased the expression of genes related to the oocyte-like phenotype. Conclusion: Our finding revealed that the combination of LIFs and estradiol could induce the GCs' oogenesis capacity and thereby is possibly suggested as a therapeutic strategy during the occurrence of gynecological disorders.

A Two-Color Haploid Genetic Screen Identifies Novel Host Factors Involved in HIV-1 Latency.

To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latent HIV-1 infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency. A select set of candidate genes was functionally validated using short hairpin RNA (shRNA)-mediated depletion in latent HIV-1 infected J-Lat A2 and 11.1 T cell lines. We confirmed ADK, CHD9, CMSS1, EVI2B, EXOSC8, FAM19A, GRIK5, IRF2BP2, NF1, and USP15 as novel host factors involved in the maintenance of HIV-1 latency. Chromatin immunoprecipitation assays indicated that CHD9, a chromodomain helicase DNA-binding protein, maintains HIV-1 latency via direct association with the HIV-1 5' long terminal repeat (LTR), and its depletion results in increased histone acetylation at the HIV-1 promoter, concomitant with HIV-1 latency reversal. FDA-approved inhibitors 5-iodotubercidin, trametinib, and topiramate, targeting ADK, NF1, and GRIK5, respectively, were characterized for their latency reversal potential. While 5-iodotubercidin exhibited significant cytotoxicity in both J-Lat and primary CD4+ T cells, trametinib reversed latency in J-Lat cells but not in latent HIV-1 infected primary CD4+ T cells. Importantly, topiramate reversed latency in cell line models, in latently infected primary CD4+ T cells, and crucially in CD4+ T cells from three people living with HIV-1 (PLWH) under suppressive antiretroviral therapy, without inducing T cell activation or significant toxicity. Thus, using an adaptation of a haploid forward genetic screen, we identified novel and druggable host factors contributing to HIV-1 latency. IMPORTANCE A reservoir of latent HIV-1 infected cells persists in the presence of combination antiretroviral therapy (cART), representing a major obstacle for viral eradication. Reactivation of the latent HIV-1 provirus is part of curative strategies which aim to promote clearance of the infected cells. Using a two-color haploid screen, we identified 69 candidate genes as latency-maintaining host factors and functionally validated a subset of 10 of those in additional T-cell-based cell line models of HIV-1 latency. We further demonstrated that CHD9 is associated with HIV-1's promoter, the 5' LTR, while this association is lost upon reactivation. Additionally, we characterized the latency reversal potential of FDA compounds targeting ADK, NF1, and GRIK5 and identify the GRIK5 inhibitor topiramate as a viable latency reversal agent with clinical potential.

Melatonin as a Therapeutic Agent for the Inhibition of Hypoxia-Induced Tumor Progression: A Description of Possible Mechanisms Involved.

Hypoxia has an important role in tumor progression via the up-regulation of growth factors and cellular adaptation genes. These changes promote cell survival, proliferation, invasion, metastasis, angiogenesis, and energy metabolism in favor of cancer development. Hypoxia also plays a central role in determining the resistance of tumors to chemotherapy. Hypoxia of the tumor microenvironment provides an opportunity to develop new therapeutic strategies that may selectively induce apoptosis of the hypoxic cancer cells. Melatonin is well known for its role in the regulation of circadian rhythms and seasonal reproduction. Numerous studies have also documented the anti-cancer properties of melatonin, including anti-proliferation, anti-angiogenesis, and apoptosis promotion. In this paper, we hypothesized that melatonin exerts anti-cancer effects by inhibiting hypoxia-induced pathways. Considering this action, co-administration of melatonin in combination with other therapeutic medications might increase the effectiveness of anti-cancer drugs. In this review, we discussed the possible signaling pathways by which melatonin inhibits hypoxia-induced cancer cell survival, invasion, migration, and metabolism, as well as tumor angiogenesis.

Crosstalk between miRNA and PI3K/AKT/mTOR signaling pathway in cancer.

Phosphoinositide-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is one of the most important proliferative signaling pathways with critical undeniable function in various aspects of cancer initiation/progression, including proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. On the other hand, numerous genetic alterations in the key genes involved in the PI3K/AKT/mTOR signaling pathway have been identified in multiple solid and hematological tumors. In addition, accumulating recent evidences have demonstrated a reciprocal interaction between this signaling pathway and microRNAs, a large group of small non-coding RNAs. Therefore, in this review, it was attempted to discuss about the interaction between key components of PI3K/AKT/mTOR signaling pathway with various miRNAs and their importance in cancer biology.

SIRT1: a promising therapeutic target in type 2 diabetes mellitus.

A significant increase in the worldwide incidence and prevalence of type 2 diabetic mellitus (T2DM) has elevated the need for studies on novel and effective therapeutic strategies. Sirtuin 1 (SIRT1) is an NAD + dependent protein deacetylase with a critical function in the regulation of glucose/lipid metabolism, insulin resistance, inflammation, oxidative stress, and mitochondrial function. SIRT1 is also involved in the regulation of insulin secretion from pancreatic ß-cells and protecting these cells from inflammation and oxidative stress-mediated tissue damages. In this regard, major SIRT1 activators have been demonstrated to exert a beneficial impact in reversing T2DM-related complications including cardiomyopathy, nephropathy, retinopathy, and neuropathy, hence treating T2DM. Therefore, an accumulating number of recent studies have investigated the efficacy of targeting SIRT1 as a therapeutic strategy in T2DM. In this review we aimed to discuss the current understanding of the physiological and biological roles of SIRT1, then its implication in the pathogenesis of T2DM, and the therapeutic potential of SIRT1 in combating T2DM.

Thymoquinone inhibited vasculogenic capacity and promoted mesenchymal-epithelial transition of human breast cancer stem cells.

BACKGROUND: Vasculogenic mimicry (VM) is characterized by the formation of tubular structure inside the tumor stroma. It has been shown that a small fraction of cancer cells, namely cancer stem cells (CSCs), could stimulate the development of vascular units in the tumor niche, leading to enhanced metastasis to the remote sites. This study aimed to study the inhibitory effect of phytocompound, Thymoquinone (TQ), on human breast MDA-MB-231 cell line via monitoring Wnt/PI3K signaling pathway. METHODS: MDA-MB-231 CSCs were incubated with different concentrations of TQ for 48 h. The viability of CSCs was determined using the MTT assay. The combination of TQ and PI3K and Wnt3a inhibitors was examined in CSCs. By using the Matrigel assay, we measured the tubulogenesis capacity. The percent of CD24- CSCs and Rhodamine 123 efflux capacity was studied using flow cytometry analysis. Protein levels of Akt, p-Akt, Wnt3a, vascular endothelial-cadherin (VE-cadherin), and matrix metalloproteinases-2 and -9 (MMP-2 and -9) were detected by western blotting. RESULTS: TQ decreased the viability of CSCs in a dose-dependent manner. The combination of TQ with PI3K and Wnt3a inhibitors reduced significantly the survival rate compared to the control group (p < 0.05). TQ could blunt the stimulatory effect of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) on CSCs (p < 0.05). The vasculogenic capacity of CSCs was reduced after being-exposed to TQ (p < 0.05). Western blotting revealed the decrease of CSCs metastasis by suppressing MMP-2 and -9. The protein level of VE-cadherin was also diminished in TQ-treated CSCs as compared to the control cell (p < 0.05), indicating inhibition of mesenchymal-endothelial transition (MendT). TQ could suppress Wnt3a and PI3K, which coincided with the reduction of the p-Akt/Akt ratio. TQ had the potential to decrease the number of CD24- CSCs and Rhodamine 123 efflux capacity after 48 h. CONCLUSION: TQ could alter the vasculogenic capacity and mesenchymal-epithelial transition of human breast CSCs in vitro. Thus TQ together with anti-angiogenic therapies may be a novel therapeutic agent in the suppression of VM in breast cancer.

The potential therapeutic effects of melatonin on breast cancer: An invasion and metastasis inhibitor.

Breast cancer is the most common cancer among women and its metastasis which generally observed at the last stage is the major cause of breast cancer-related death. Therefore, the agents that have the potential to prevent metastatic and invasive nature of breast cancer can open up new therapeutic strategies. Melatonin, a major hormone of pineal gland, is a powerful anti-cancer agent. There are growing evidence regarding the protective effect of melatonin against cancer invasion and metastasis. The anti-metastatic feature of melatonin accompanies with suppression of tumor proliferation, induction of tumor apoptosis, regulation of the cell cycle, modulating angiogenesis, impediment of invasion, and induction of cancer cells sensitivity to the chemotherapy agents. More recently, anti-metastatic effect of melatonin through affecting cancer stem cells and vascular mimicry has been identified. Thus, the aim of this review is to discuss the potential therapeutic effect of melatonin on breast cancer via modulating the cells invasion and metastasis.

Targeting Notch signaling pathway as an effective strategy in overcoming drug resistance in ovarian cancer.

Ovarian cancer, as one of the most common types of gynecological malignancies, has an increasing rate of incidence worldwide. Despite huge amounts of recent efforts in designing novel therapeutic strategies for complete removal of tumors and increasing overall survival of patients, chemotherapy is still the preferred therapy for ovarian cancer. However, chemotherapy is also challenged by development of drug resistance. Therefore, elucidating the underlying mechanisms of drug reissuance is an urgent need in ovarian cancer. Numerous studies have shown the implication of the Notch signaling pathway in the development of various human malignancies. Therefore, this study will provide a brief overview of the published evidence in support of Notch targeting in reverting multidrug resistance as a safer and novel approach for the improvement of ovarian cancer treatment.

Inhibitory effect of melatonin on hypoxia-induced vasculogenic mimicry via suppressing epithelial-mesenchymal transition (EMT) in breast cancer stem cells.

Vasculogenic mimicry (VM) play an important role in breast cancer metastasis and anti- angiogenic drugs resistance. Hypoxia, the epithelial-mesenchymal transition (EMT), and cancer stem cells (CSCs) are known as essential factors for VM formation. Also, melatonin is an amino acid-derived hormone with many anti-tumor effects. Despite the antitumor effects of melatonin, its effect on VM formation in breast cancer has not been considered yet, so we investigated the effect of melatonin on VM formation through EMT process under hypoxia conditions in breast CSCs. The CSCs percentage and VM formation were determined in MCF-7 and MDA-MB-231, respectively. Also, analysis of HIF-1α expression under hypoxia in MDA-MB-231 and MCF-7 cell lines was performed using Western blot. The effect of melatonin on the VM formation, invasion, and migration was also investigated. Moreover, the effect of melatonin on the expression EMT markers was evaluated. CD44+ CD24-phenotype as CSCs marker in MDA-MB-231 cell line, was 80.8%, while it was 11.1% in MCF-7 cell line. HIF-1α expression was up-regulated in the VM-positive breast cancer cell line MDA-MB-231, and consequently, affected the expression of the EMT markers E-cadherin, vimentin, snail, and MMP9. Melatonin had significant effect on EMT and formations of VM in breast CSCs. Melatonin could prevent the formation of VM by affecting the important molecules involved in the formation of VM structures and the EMT. Moreover, our data clearly showed that, melatonin represents molecule with significant anti-cancer activities that may potentially optimize the management of breast cancer through the overcoming drug resistance in anti-angiogenic drugs.