The RNAi enhancer enoxacin inhibits the growth and migration of esophageal squamous cell carcinoma cells

The RNAi enhancer enoxacin inhibits the growth and migration of esophageal squamous cell carcinoma cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The RNAi enhancer enoxacin inhibits the growth and migration of esophageal squamous cell carcinoma cells Parisa Torabi, Hanieh Torkian, Seyed Rohullah Miri, Sharif Moradi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4146187/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. A decrease in the global expression of microRNAs (miRNAs) is observed in various types of cancer, including esophageal cancer. It has been found that the small molecule enoxacin serves as an RNA interference (RNAi) enhancer, increasing the maturation rate of various cellular miRNAs. Here, we show that enoxacin significantly reduces the growth characteristics of ESCC cell lines. It induces cell cycle arrest and apoptosis in ESCC cells, leading to a clear decrease in ESCC cell number and viability. In addition, enoxacin suppresses the ability of cells to migrate and decreases their capacity to form colonies. Mechanistically, we reveal that enoxacin promotes the maturation of miRNAs through the stimulation of TARBP2 protein, the physical partner of DICER1. Taken together, enoxacin potently blocks the growth, motility, and clonogenicity of ESCC cells, paving the way for further investigation of this small-molecule chemical in animal models of ESCC. Biological sciences/Cancer/Cancer therapy Biological sciences/Cancer/Gastrointestinal cancer Biological sciences/Biotechnology/Oligo delivery Biological sciences/Biological techniques/Gene expression analysis Biological sciences/Biological techniques/Molecular engineering oncomiR tumorigenicity esophagus fluoroquinolone malignancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Esophageal cancer is one of the deadliest and most aggressive types of cancer worldwide 1 . It has an extremely poor prognosis, and a 5-year survival rate of 15–25% 2 . There are two main histopathological types of esophageal cancer including esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) with the latter being the most prevalent form of esophageal cancer worldwide, accounting for more than 80% of all cases in some Asian countries such as China and Iran 3 . Although the currently available therapeutic approaches (i.e. curative surgery, chemotherapy, and radiotherapy) to ESCC have improved, the majority of these treatment strategies are not sufficiently effective, resulting in a marginal improvement in survival rates over time. Therefore, new therapeutic tools are needed to enable more effective treatment of ESCC patients 4 . MicroRNAs (miRNAs) are a class of small, non-coding RNAs that post-transcriptionally regulate gene expression 5 . There have been numerous reports on the occurrence of mutations and disruptions in proteins, such as DICER, DROSHA, and TARBP2, which are directly involved in the biogenesis pathway of miRNAs in a variety of human cancers, including esophageal cancer. Such deregulations result in a global reduction in the expression of virtually all cellular miRNAs 6 . Alterations in the cellular miRNAome often cause major changes in the cellular proteome and consequently in the cell behavior 7,8 . Notably, global reductions in miRNA expression resulting from deregulations in miRNA biogenesis genes have been frequently observed to be associated with development and enhanced malignancy of several types of cancer including esophageal cancer 9,10 . Hence, stimulating the miRNA biogenesis pathway in esophageal cancer might be a viable option for treating the development and progression of this less-survivable disease. The fluoroquinolone family of antibiotics has been demonstrated to suppress the growth and invasiveness of multiple types of tumor cells without causing notable adverse effects on normal cells 11 . As shown in Table 1, in vitro and in vivo investigations on various types of cancer using several members of this family of antibiotics have shown a significant inhibitory effect on cancer cell growth and proliferation. Among all fluoroquinolones tested, enoxacin has frequently been found to have the most potent inhibitory effect on cancer cells 12,13 . It was revealed that the anti-cancer effects of enoxacin is exerted through the general enhancement of the RNA interference (RNAi) pathway, thereby stimulating the miRNA biogenesis pathway 14 . Enoxacin was then discovered to directly bind to the DICER’s physical partner TARBP2 and stimulate its protein activity leading to a general increase in the expression of cellular miRNAs which could bring the pattern of miRNA expression in cancer cells closer to that in the normal cells 15,16 . Based on these findings, we hypothesized that the RNAi enhancer enoxacin might be able to inhibit the viability and other growth features of ESCC cells in vitro. Our analysis revealed that enoxacin reproducibly suppressed the survival of ESCC cells compared to the control groups. It also significantly reduced the cell cycling as well as colony formation ability of the cells. Moreover, the migration of the ESCC cells was clearly reduced upon treatment with enoxacin. Finally, we found that enoxacin modulated the expression of several cancer-associated miRNAs through the simulation of the TARBP2 protein. Mechanistically, antisense oligonucleotides (ASOs) targeting TARBP2 transcript neutralized the inhibitory effects of enoxacin on ESCC cells, indicating that enoxacin exerts its anti-tumor effects on ESCC cells through stimulating TARBP2 activity. Overall, we report that globally enhancing the RNAi pathway leaves a potent inhibitory impact on ESCC cells. Methods Cell lines and cell culture ESCC cell lines (KYSE-30 and YM-1) and human foreskin fibroblast (HFF) cells were cultured in respective basal media, i.e. RPMI 1640 (Gibco) for KYSE-30, DMEM F12 (Gibco) for YM-1, or high-glucose DMEM (Gibco) for HFF cells, supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Invitrogen), 0.1% mM β-mercaptoethanol (Sigma-Aldrich), 1% Glutamax (Invitrogen), and 1% non-essential amino acids (Invitrogen). KYSE-30 cell line was a gift from Dr. Seyed-Javad Mowla lab at Tarbiat Modares University, Tehran, Iran. YM-1 cell line was a gift from Dr. Jahanbakhsh Asadi lab at Golestan University of Medical Sciences, Gorgan, Iran. Cells were maintained at 37°C in a humidified incubator containing 5% CO 2 and cultured in T-25 vessels and other tissue-culture dishes (TPP). All cell lines were regularly tested for mycoplasma contamination using Hoechst staining and specific PCR Mycoplasma Detection kits. The media of the cells were changed every other day. Enoxacin treatment Enoxacin (Sigma-Aldrich) saline solution was prepared in water and stored at 4°C until further use. Enoxacin treatment was performed by adding the saline solution with a concentration of 124 µM to the culture medium. Due to the solubility of the salt form of enoxacin in water, no special substance or solvent was added to the culture medium in the control groups. Enoxacin has a limited water solubility and must be properly pipetted before use. Transfection of TARBP2 antisense oligonucleotides The oligonucleotides used in this study were synthesized by miRas Biotech (Tehran, Iran). TARBP2 -specific ASOs and scrambled (Scr) control oligonucleotides were delivered into the cells at a final concentration of 100 nM using DharmaFECT1 reagent (Dharmacon). The cells were cultured in culture dishes 24 h prior to transfection. Before doing transfections, the oligonucleotides as well as the DharmaFECT1 reagent were separately diluted in serum-free DMEM/F-12 media, the diluted oligonucleotides and the reagent were mixed at a 1:1 ratio, and then the resulting complex was kept at room temperature for 20 min. After oligonucleotide transfection, the cells were maintained in a 5% CO2 humidified incubator. The experiments were carried out in triplicate and the data were shown as mean ± SD. 18 h post-transfection, the media of the cells were refreshed to remove the transfection reagent. Cells were analyzed 5 days post-treatment. Viability assays MTS assay Cell viability was evaluated using the MTS assay. In brief, 2.0 × 10 3 cells were seeded in 96-well cell culture plates and allowed to adhere overnight. Cell viability was measured 3 days post-treatment. To measure cell viability, the MTS solution was added to cell media in a 1:10 dilution ratio. The cells were then incubated at 37°C and 5% CO 2 until a significant color change was observed (typically within 3 h). Finally, cell viability was measured by determining the absorbance at 495 nm using a microplate reader (ThermoFisher Scientific). The results were reported as mean ± SD, p value ≤ 0.05 in 3 biological replicates. Live-dead assay To perform live-dead assay, the cell supernatant was removed 3 days post-treatment, the cells were washed with PBS − , and the dye solution of the live-dead assay was added to the cells. The dye solution, which is stored at 4°C and in dark before use, consists of calcine methyl ester (Calcein AM) and ethidium homodimer in a ratio of 1:5 in PBS − . Cells were incubated in the presence of the dye solution for 20 min at 37°C, and were then viewed and imaged using fluorescence microscopy. In this assay, the green cells are live, the red cells are dead, and the yellow cells are dying cells. Crystal violet staining The cells were fixed with 4% paraformaldehyde solution at room temperature for 20 min, then washed with PBS − . The crystal violet dye solution was diluted 1:50 in PBS − and placed at room temperature. After 5 min, the cells were washed with PBS − and visualized under a phase-contrast microscope. Crystal violet dye solution is prepared by dissolving 5 mg of the dye powder in 1 ml of 20% ethanol solution. Cell cycle analysis Three days following treatment, 1.0 × 10 6 cells were transferred to flow cytometry tubes and pelleted using centrifugation (5 min, 15000 rpm). After washing cell pellets 3 times with PBS, the nuclear DNA was fixed by adding 70% ice-cold ethanol is a dropwise manner along with low-speed vortexing followed by the incubation of the cells at 4°C for 2 h. Then, the cell pellet was washed twice with PBS − to remove ethanol. In order to deactivate RNAs, 100 µl of RNase I was added to the cell pellet and incubated at 37°C for 20 min. Then, the cells were placed ice to inactivate the RNase enzyme. Cell cycle profiles were determined using flow cytometry with a FACSCalibur II instrument. Investigation of apoptosis by flow cytometry Cells and their supernatant were collected separately 3 days post-treatment. The cells were then subjected to annexin V (Santa Cruz) and propidium iodide (PI, Sigma-Aldrich) staining using FITC Annexin V Apoptosis Detection Kit 1 (BD Biosciences). The rate of cell apoptosis was measured within 30 min post-staining using flow cytometry with a FACSCalibur II instrument. The data of early and late apoptosis and necrotic cells were analyzed by FlowJo software. Colony formation assay To analyze the colony formation ability of the cells, the cells were treated with enoxacin for 3 days, respectively. On day 3, the cells were trypsinized of which 200 cells were seeded into 35-mm dishes. The cells were then allowed to grow for 3 weeks to form colonies. Next, the colonies were stained with the crystal-violet dye solution, and the type and number of colonies were examined under a phase-contrast microscope. Wound healing assay To investigate potential changes in the migratory behavior of the cells upon treatment, 5 × 10 5 cells were cultured in each well of 12-well plates and treated for 3 days after reaching to ~ 90% cell density. To attribute any changes in the area of the scratch to cell migration only, cell proliferation was inhibited by 1% mitomycin treatment for 18 h. Then, a scratch was made on the cells with the help of a yellow pipette tip under a loop microscope and the cells were then washed several times with PBS − . Images were taken every 24 h for 3 days. RNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) Total RNA extraction was performed using Qiagen’s miRNeasy mini kit, and RNA concentration and purity were measured using a spectrophotometer. The cDNA was synthesized using Thermo Scientific™ RevertAid RT Reverse Transcription Kit for mRNA and Exiqon’s miRCURY LNA RT Kit for miRNA. The resulting single-stranded cDNA was stored at -80° C for long-term storage. PCR quantification of expression levels of gene transcripts was carried out using high ROX Takara Bio Green Premix Ex Taq II. The expression levels were normalized against the expression level of the endogenous control gene transcript GAPDH . qRT-PCR analysis of miRNA was performed using SYBR green-based miRCURY LNA™ Universal RT microRNA PCR system and LNA enhanced miRNA specific primers (Exiqon) according to the manufacturer’s instructions. miRNA expression levels were normalized against the small RNA endogenous control U 6 snRNA. Statistical analysis The results obtained in this study were analyzed using t test and one-way or two-way ANOVA depending on the number of variables by GraphPad Prism 9.0.0 software. Experiments were performed with at least 3 biological and 2 technical replicates. The data are presented as mean ± SD and the significance of the data was represented as * p value < 0.05, ** p value < 0.01, *** p value < 0.001, and **** p value < 0.0001. Results Enoxacin has a cancer-specific growth-inhibitory effect on ESCC cells To analyze the effects of enoxacin on ESCC cells, we first asked if enoxacin could reduce ESCC cell viability. The initial step was to determine the optimized enoxacin concentration to treat the cells. To this end, we cultured ESCC cells in 96-well plates, treated them for 5 days with different concentrations of enoxacin (0, 10, 25, 50, 75, 100, 125, 150, 200, 250, 300, and 350 µM), and measured ESCC cell viability using MTS assays. We observed that enoxacin dose-dependently suppressed the growth of ESCC cells, and determined its median inhibitory concentration (IC 50 ) to be 125 µM (Supplementary Fig. 1a). Since several other studies have determined the IC 50 of enoxacin to be 124 µM 15,17 , we decided to perform all our subsequent treatments with 124 µM which is almost identical to the IC 50 that we determined in the current study. Notably, this inhibitory effect was observed to be cancer-specific, as enoxacin did not adversely affect the growth of non-cancerous cells i.e. human foreskin fibroblasts (HFFs), judging from crystal violet staining, MTS viability assessments, and live/dead staining (Supplementary Fig. S1 b-d). We subsequently treated the ESCC cells with 124 µM of enoxacin continuously for 5 days for various experiments, as the 5-day treatment duration exerted a more potent inhibition than 3- or 4-day treatments on cell viability (Supplementary Fig. 1e). We also noticed that it was not necessary to treat the cells daily with fresh enoxacin as there was not a significant difference in cell viability when changing the media either every 24 h or every 48 h (Supplementary Fig. 1f). Therefore, we exposed the ESCC cells to enoxacin for 5 continuous days and refreshed the media of the cells every 48 h for the subsequent assays. Next, the effect of enoxacin on growth and survival of the ESCC cell line, KYSE-30, was investigated. The cells were exposed to enoxacin for 5 days, and their survival was measured on day 5. We found that the number and viability of KYSE-30 cells were considerably decreased by enoxacin treatment compared to the untreated group, as evidenced by phase-contrast imaging (Fig. 1 a), crystal violet staining (Fig. 1 b), and live/dead assays where enoxacin decreased the number of live (green) cells and simultaneously increased the number of dead (red) and dying (yellow) cells (Fig. 1 c-d). This result was further confirmed using MTS viability assays (Fig. 1 e) and the analysis of cell growth rate on the basis of cell counting following enoxacin treatment (Fig. 1 f). We next repeated the same experiments in a second ESCC cell line, YM-1, to examine how reproducible the growth-inhibitory effects of enoxacin are. We obtained a highly-similar set of results for YM-1 cells and indicated that enoxacin potently inhibited the growth of YM-1 cells judging from phase-contrast imaging (Fig. 2 a), crystal violet staining (Fig. 2 b), live/dead staining (Fig. 2 c-d), MTS viability assays (Fig. 2 e), and cell counting (Fig. 2 f). These results indicate that enoxacin reproducibly inhibits the survival of ESCC cells whereas it has no inhibitory effects on the viability of normal cells. ESCC cells undergo cell cycle arrest and apoptosis upon treatment with enoxacin After noticing a clear decline in survival of ESCC cells while being exposed to enoxacin, we looked at the cell cycle and apoptosis rate of the cells to examine if these two phenomena contribute to the decreased cell number observed upon enoxacin treatment. To determine the cell cycle profile, ESCC cells were seeded in 35-mm culture dishes, and were synchronized in G1 phase by being maintained under FBS-free conditions before the treatment. We found that 5 days treatment with enoxacin resulted in cell cycle arrest in the S phase of the KYSE-30 cell cycle (Fig. 3 a-b) and in the G1 phase of YM-1 cell cycle (Fig. 3 c-d), indicating that enoxacin impairs the cell cycling of the cancer cells. Next, we sought to determine the apoptotic rate of the cells following exposure to enoxacin. Four days post-treatment, the KYSE-30 cells in the treatment and control groups were harvested and prepared for annexin-PI staining. Flow cytometry analysis of the cells stained with annexin-PI revealed that in the enoxacin-treated cells compared to the control group, (i) the number of live (non-apoptotic) cells was lower and (ii) the percentage of cells in the late phase of apoptosis was significantly higher (Fig. 3 e-f). We also observed a similar increase in apoptosis rate when exposing YM-1 cells to enoxacin (Fig. 3 g-h). Overall, enoxacin treatment induces cell cycle arrest as well as apoptosis in ESCC cells, thereby explaining the observed reductions in the number of ESCC cells upon treatment with the chemical. Enoxacin inhibits capacity of ESCC cells to form colonies and migrate Next, we sought to determine whether enoxacin affects the clonogenicity of ESCC cells. To this end, the cells were treated with enoxacin for 5 days, and on day 5, the cells in both control and treatment groups were trypsinized, from which 200 cells were transferred to 35-mm cell culture dishes containing complete ESCC medium for colony formation. Within 3 weeks, several colonies emerged from the seeded cells which were then stained on day 21 with crystal violet solution and counted. The results indicated that enoxacin efficiently inhibited colony formation of KYSE-30 cells (Fig. 4 a). The number and type of colonies were then analyzed and it was revealed that the colonies of the control group cells were mostly holoclones with a few colonies being meroclones or paraclones (Fig. 4 b-c). In contrast, the colonies derived from the enoxacin-treated cells were more similar to cell clusters than cell colonies with few of them being paraclones (i.e. no holoclones and meroclones observed) (Fig. 4 b-c). We also studied the YM-1 cell line in terms of clonogenicity in response to enoxacin exposure, and found a clear reduction in the number of colonies formed in the treated group compared to the control group (Fig. 4 d-f), confirming that enoxacin is reproducibly detrimental to colony formation by ESCC cells. Next, we asked if enoxacin inhibits the ability of cancer cells to migrate. Toward this goal, we carried out wound healing (i.e. scratch) assays post-treatment and examined the scratch area over 24 h. As shown in Fig. 5 a-b, enoxacin considerably inhibited the closure of scratch wounds of the cultured cancer cells compared to the untreated cells. Since cancer cell migration is known to be dependent on cancer stem cells, we then analyzed the expression of several stemness genes e.g. OCT4 , SOX2 , NANOG , KLF4 , c-MYC , and LIN28 upon treatment with enoxacin. Our qRT-PCR analysis revealed that enoxacin suppressed the expression of almost all these genes 48 h post-treatment compared to the untreated control group (Fig. 5 c). Moreover, it significantly decreased the expression of typical cancer-associated genes ALDH1 , CD44 , and CD133 (Fig. 5 d). In conclusion, enoxacin blocks ESCC cell migration by downregulating key stemness and cancer genes. TARBP2 mediates the anti-tumor effects of enoxacin in ESCC cells Previous research has demonstrated that enoxacin induces cancer cell suppression by speeding up the maturation of miRNAs through binding and stimulating the TARBP2 protein, which is the physical partner of DICER1. To examine if enoxacin similarly promotes TARBP2-mediated processing of cellular miRNAs in ESCC cells, we treated the cancer cells with enoxacin and analyzed the expression of a selected set of miRNAs 48 h after exposure. It was observed using qRT-PCR assays that all the tested miRNAs were upregulated in response to enoxacin treatment (Fig. 6 a), which was consistent with what reported previously 15 . Next, we attempted to confirm whether TARBP2 is the mediator of enoxacin actions in ESCC cells. To this end, we first transfected the cells with a FITC-conjugated oligonucleotide to determine the efficiency of our transfection-based approach for the delivery of the TARBP2 -targeting ASOs. We observed that our lipid-based transient transfection method delivers the FITC-labelled oligonucleotides to the cultured ESCC cells in a highly efficient manner (~ 70% of the cells received the oligonucleotide), as evidenced by fluorescence microscopy and flow cytometry (Supplementary Fig. 2a-b). Next, we suppressed the expression of TARBP2 transcript using a pool of two different TARBP2 -targeting ASOs with two different concentrations to determine the optimal ASO concentration for TARBP2 silencing, and found that a significant downregulation of TARBP2 transcripts could be similarly achieved upon treatment with either of the ASO concentrations (Fig. 6 b), therefore we chose the lower concentration (100 nM) for transfection. Then, we treated the ESCC cells according to the procedure shown in Fig. 6 c to examine if TARBP2 knockdown abolishes the growth-inhibitory effects of enoxacin. As shown in Fig. 6 d-i, (i) enoxacin alone sharply reduced cell viability; (ii) TARBP2 -targeting ASOs had no inhibitory effects on ESCC cell growth; and (iii) enoxacin-ASO combination group did not reduce cell viability as potent as enoxacin alone, judging from phase-contrast imaging (Fig. 6 d), crystal violet staining (Fig. 6 e), live/dead staining (Fig. 6 f-g), cell counting (Fig. 6 h), and MTS viability assays (Fig. 6 i). Taken together, these results confirm that enoxacin suppresses the growth of ESCC cells by TARBP2 -mediated enhancement of miRNA maturation. Discussion Cancer is the second leading cause of death worldwide. Esophageal cancer is one of the deadliest malignancies and less survivable cancers which is due to sub-optimal diagnostic tools and therapeutic strategies for the disease 18 . Iran and East Asia along with some Western societies have exceptionally high rates of this cancer type 19 . The majority of human cancers are known to have inactivating mutations in key components of the miRNA biogenesis pathway such as DROSHA, TARBP2, and DICER1, and exhibit a global downregulation in mature miRNA levels 20 . This suggests that not only cells’ miRNAomes generally play a crucially important anti-cancer role 8,21–23 , but also restoring a normal cell-like miRNA profile to cancer cells may be therapeutically-viable approach 8,24 . Therefore, agents that are able to globally enhance the RNAi (siRNA/miRNA biogenesis) pathway may serve anti-cancer functions. Enoxacin is a member of the fluoroquinolone family of antibiotics that are frequently used to treat bacterial infections of the urogenital tract. In 2008, enoxacin was found to also influence the human (eukaryotic) cells by binding to TARBP2, the physical partner of DICER1 and potently enhancing the RNAi pathway 14,25,26 . It was later found that enoxacin’s stimulatory effect on the miRNA pathway promotes apoptosis in several types of cancer cells 15,27,28 . Since miRNAs play important roles in esophageal cancer cells 29–31 , we hypothesized that enoxacin might inhibit these cells via enhancing the RNAi pathway. We observed that enoxacin treatment markedly reduced cell survival, ability to proliferation, colony formation, and migration ability of ESCC cells, but had no negative effects on the viability of normal cells as expected, because the drug had previously been approved by several international food and drug administrations to be safe for use in humans as an antibiotic. Notably, certain fluoroquinolones such as ciprofloxacin are currently used in the cancer treatment protocols to prevent or reduce the risk of wound infection following tumor tissue surgery 32–34 , and in this sense, enoxacin may play a dual role and simultaneously serve as an anti-cancer and inti-infection drug for cancer patients. Mechanistically, we showed that enoxacin upregulated several randomly chosen miRNAs that already show expression in ESCC cells. To further corroborate the mechanism of enoxacin action, we silenced its major target protein TARBP2 using specific ASOs and found that enoxacin no longer inhibited the growth of ESCC cells upon TARBP2 knockdown. This result confirmed that enoxacin acts through TARBP2 to drive ESCC cell death. It would be worthwhile to analyze whether enoxacin could similarly inhibit the growth of ESCC cells in animal models of esophageal cancer. In summary, we showed that enoxacin considerably reduced the survival, cell cycling, and motility of esophageal cancer cells (KYSE-30 and YM-1 cell lines). Several key molecular players involved in the maintenance and progression of cancer stemness and malignancy of ESCC were observed to be suppressed by enoxacin. Moreover, we validated that enoxacin exerts its anti-tumor effects by stimulating TARBP2, thereby upregulating several tumor suppressing miRNAs. It is of critical importance to investigate the effect of enoxacin alone as well as in combination with other anti-cancer agents in animal models of ESCC (in vivo). Importantly, fluoroquinolones including enoxacin are small-molecule chemicals that can be taken orally and are known to exhibit a high bioavailability upon consumption by the patients. Although enoxacin is a small molecule that efficiently diffuses into the various tissues of the body, it may still be necessary to analyze the accessibility of the tumor cells to this drug. This is because under hypoxic conditions in the core of tumors, most of the cells located in this area are cancer stem cells and receive smaller concentrations of the drugs due to their less-developed vasculature. Cancer stem cells are resistant to various treatment regimens, therefore it would be important to investigate the exposure of ESCC cancer stem cells to enoxacin and examine how the stem cells respond. Further research would be required to reveal if enoxacin would have the potential to be used in humans as an anti-cancer drug. Declarations Acknowledgements We would like to thank members of Moradi lab for critical discussions. This work was supported by a grant from Iran National Science Foundation (INSF) to Sharif Moradi (grant No. 98013813). Author Contributions PT and HT performed the experiments, analyzed the data, and contributed to drafting of the manuscript. SRM served as scientific adviser. SM conceptualized, supervised, provided financial support, and wrote and approved the manuscript for submission. Data Availability Statement All data supporting the findings of this study are available in the article and the associated supplementary file. Ethical Approval Not applicable. 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Scientific Reports 7 , 43379 (2017). Vracar, T. C. et al. Enoxacin and bis-enoxacin stimulate 4T1 murine breast cancer cells to release extracellular vesicles that inhibit osteoclastogenesis. Scientific reports 8 , 16182 (2018). McDonnell, A. M., Pyles, H. M., Diaz-Cruz, E. S. & Barton, C. E. Enoxacin and epigallocatechin gallate (EGCG) act synergistically to inhibit the growth of cervical cancer cells in culture. Molecules 24 , 1580 (2019). Additional Declarations Competing interest reported. SM is the CEO of miRas Biotech. miRas Biotech had no role in designing or analyzing the experiments. Other authors have no competing interests. Supplementary Files SupplementaryMaterialTorabietal.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4146187","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":288498283,"identity":"e107b556-09ba-4354-a724-698415b5eb38","order_by":0,"name":"Parisa Torabi","email":"","orcid":"","institution":"Royan Institute","correspondingAuthor":false,"prefix":"","firstName":"Parisa","middleName":"","lastName":"Torabi","suffix":""},{"id":288498284,"identity":"d54aa380-9b9d-4fb7-84f4-5cab0eca6213","order_by":1,"name":"Hanieh Torkian","email":"","orcid":"","institution":"Royan Institute","correspondingAuthor":false,"prefix":"","firstName":"Hanieh","middleName":"","lastName":"Torkian","suffix":""},{"id":288498285,"identity":"b0aa55a7-5512-407c-827c-3221ecdfed27","order_by":2,"name":"Seyed Rohullah Miri","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Rohullah","lastName":"Miri","suffix":""},{"id":288498286,"identity":"7fe9d7a2-aba5-4505-927a-31c015ae5ea8","order_by":3,"name":"Sharif Moradi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYDACCQjF2MbegE0crxaeA6RqaZBIINJd8rMbWDd83GMn2yf59uFjnpo7DObtBxg//GCwyMelxeDOAbabM54lG7dJpxsb8xx7xiBzJoFZsodBwrIBlxaJBLbbPAeYE9uk09ikedgO188AOlUa6GADnA6bAdZSn9gmeQyo5d9hBgkJBubf+LQw3ABrOZzYJsHGJs3bBtbChtcWgxuJbTdnHDhu3MaTxmw4tw+ohSexzbLHAJ/Dko/d+HCgWnZ++zHGB2++AbWwHz5840dFHW6HAWMEzmTigYvg0YCq+weRCkfBKBgFo2BkAQCSLkxu8/axGQAAAABJRU5ErkJggg==","orcid":"","institution":"Royan Institute","correspondingAuthor":true,"prefix":"","firstName":"Sharif","middleName":"","lastName":"Moradi","suffix":""}],"badges":[],"createdAt":"2024-03-22 00:44:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4146187/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4146187/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54307004,"identity":"9006178c-310d-40da-bc29-b21dc834ceb3","added_by":"auto","created_at":"2024-04-08 15:43:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1016457,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnoxacin has a cancer-inhibitory effect in KYSE-30 ESCC cells.\u003c/strong\u003e (\u003cstrong\u003ea)\u003c/strong\u003e Phase-contrast imaging of the ESCC cell line KYSE-30 in the presence or absence of enoxacin. (\u003cstrong\u003eb)\u003c/strong\u003e Crystal violet staining of KYSE-30 cells treated with or without enoxacin. \u003cstrong\u003e(c)\u003c/strong\u003eLive-dead staining of KYSE-30 cells in the presence or absence of enoxacin. \u003cstrong\u003e(d)\u003c/strong\u003eQuantified graph of live-dead staining shown in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003e(e) \u003c/strong\u003eMTS assay of KYSE-30 cells in the presence or absence of enoxacin. \u003cstrong\u003e(f)\u003c/strong\u003e Growth curve of ESCC cells treated with or without enoxacin obtained by hemocytometer cell counting. Data are shown as mean ± SD with 4 independent replicates. **\u003cem\u003ep\u003c/em\u003evalue \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/cf2a3ad596c5bd2f041889c8.jpg"},{"id":54307003,"identity":"802f0de2-0f83-4851-8985-fba9513ac2d3","added_by":"auto","created_at":"2024-04-08 15:43:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":972481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the effect of enoxacin treatment on the growth of YM-1 cell line. (a)\u003c/strong\u003e Phase-contrast microscopic pictures of the ESCC cell line YM-1 treated with enoxacin compared to the untreated control cells. \u003cstrong\u003e(b)\u003c/strong\u003e Crystal violet staining of YM-1 cells in the presence or absence of enoxacin. \u003cstrong\u003e(c)\u003c/strong\u003eLive/dead-stained YM-1 cells exposed to enoxacin as visualized under a fluorescent microscope. \u003cstrong\u003e(d)\u003c/strong\u003e Quantification of the live-dead staining data shown in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003e(e)\u003c/strong\u003e MTS viability assessment of YM-1 cells treated with or without enoxacin. \u003cstrong\u003e(f)\u003c/strong\u003e Growth curve of YM-1 ESCC cells in the presence or absence of enoxacin obtained by hemocytometer cell counting. Data are shown as mean ± SD with four independent replicates. **\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/818f1fd1cbf8bb9600ddbdd5.jpg"},{"id":54307008,"identity":"38158bb7-bf57-42b9-bb7a-e8d803d0d7a1","added_by":"auto","created_at":"2024-04-08 15:43:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":517222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of enoxacin treatment on cell cycle profile and apoptosis of ESCC cells. (a) \u003c/strong\u003eCell cycle profile of KYSE-30 cells treated with or without enoxacin. \u003cstrong\u003e(b)\u003c/strong\u003eQuantification of the cell cycle profiling data shown in \u003cstrong\u003ea\u003c/strong\u003e. \u003cstrong\u003e(c) \u003c/strong\u003eCell cycle profiles of YM-1 cells in the presence or absence of enoxacin.\u003cstrong\u003e (d) \u003c/strong\u003eQuantification of the cell cycle profiling data shown in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003e(e)\u003c/strong\u003e Annexin V/PI staining of the KYSE-30 cells to determine their apoptosis rate following treatment with enoxacin. \u003cstrong\u003e(f) \u003c/strong\u003eQuantification of the apoptosis data shown in \u003cstrong\u003ee\u003c/strong\u003e.\u003cstrong\u003e (g) \u003c/strong\u003eAnalysis of apoptosis using annexin/PI staining followed by flow cytometry in enoxacin-treated YM-1 cells compared to the untreated control cells. \u003cstrong\u003e(h) \u003c/strong\u003eQuantified chart of apoptosis data shown in \u003cstrong\u003eg\u003c/strong\u003e. The experiments were performed in three independent biological replicates. The data is shown as mean ± SD. **\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/b049040dd7e835dc0a7f8645.jpg"},{"id":54307005,"identity":"4711a8d9-5133-44d1-84e9-6a6397ed4e1b","added_by":"auto","created_at":"2024-04-08 15:43:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":778858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of enoxacin on the colony-formation ability of ESCC cells.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Colony formation assay in KYSE-30 cells treated with or without enoxacin following crystal violet staining. \u003cstrong\u003e(b)\u003c/strong\u003e Different types of crystal violet stained colonies in the treatment (Enx) and control (ctrl) groups. \u003cstrong\u003e(c)\u003c/strong\u003e Quantified graph of the 3 types of colonies shown in \u003cstrong\u003ea \u0026amp; b\u003c/strong\u003e. \u003cstrong\u003e(d)\u003c/strong\u003e Full-dish view of YM-1 cell colonies formed from 200 YM-1 single cells 3 weeks after trypsinization of the cells pre-treated with enoxacin for 3 days followed by crystal violet staining. \u003cstrong\u003e(e)\u003c/strong\u003e Types of obtained colonies shown in \u003cstrong\u003ed\u003c/strong\u003e. \u003cstrong\u003e(f)\u003c/strong\u003e Quantification of the colony formation assay data shown in \u003cstrong\u003ed \u0026amp; e\u003c/strong\u003e. The experiments were performed in 3 independent biological replicates. The data is shown as mean ± SD. ***\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/d7d0da19de46ddeaeb11ff8e.jpg"},{"id":54307427,"identity":"88dd5d3d-7941-441a-81e9-ed6adfc29e09","added_by":"auto","created_at":"2024-04-08 15:51:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1081567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnoxacin inhibits the migration of ESCC cells. (a)\u003c/strong\u003e The effect of enoxacin on the migration ability of the cancer cells at 0, 18, and 24 h after performing the scratch. \u003cstrong\u003e(b)\u003c/strong\u003e Quantification of the results shown in \u003cstrong\u003ea\u003c/strong\u003e. (\u003cstrong\u003ec \u0026amp; d)\u003c/strong\u003e Expression analysis of \u003cstrong\u003e(c)\u003c/strong\u003e stemness- and \u003cstrong\u003e(d)\u003c/strong\u003ecancer-associated genes in cancer cells upon treatment with enoxacin. Data are expressed as mean ± SD of 3 independent replicates. **\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.01, ****\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/e2c9c97c4e609fa4c86d8983.jpg"},{"id":54307010,"identity":"c29e6775-38c8-43a9-961d-b45fe67779c4","added_by":"auto","created_at":"2024-04-08 15:43:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1253694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe miRNA biogenesis pathway mediates the effects of enoxacin treatment on ESCC cells.\u003c/strong\u003e \u003cstrong\u003e(a)\u003c/strong\u003e Effect of enoxacin on the expression of select miRNAs in ESCC cells. \u003cstrong\u003e(b)\u003c/strong\u003e qRT-PCR-based expression analysis of \u003cem\u003eTARBP2 \u003c/em\u003etranscripts 48 h following treatment with specific ASOs. \u003cstrong\u003e(c)\u003c/strong\u003e The procedure used to investigate the mechanism of enoxacin action through ASO-mediated inhibition of \u003cem\u003eTARBP2\u003c/em\u003e. \u003cstrong\u003e(d)\u003c/strong\u003e Phase contrast imaging, \u003cstrong\u003e(e)\u003c/strong\u003e crystal violet staining, and \u003cstrong\u003e(f \u0026amp; g)\u003c/strong\u003e live-dead staining of ESCC cells on day 6 according to the treatment procedure shown in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003e(h)\u003c/strong\u003e Growth status of ESCC cells obtained by hemocytometer cell counting on day 6 of the treatment procedure shown in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003e(i)\u003c/strong\u003e MTS viability assay on day 6. Data is shown as mean ± SD of 3 replicates. *\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e value \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/fcef69b910377ddd6d8b422b.jpg"},{"id":59488583,"identity":"a58e667d-fa09-4ad5-8213-f46ee0be9ecf","added_by":"auto","created_at":"2024-07-02 11:37:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6420985,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/9cfcac05-303e-46ad-8a3e-a0d9d8821bff.pdf"},{"id":54307425,"identity":"e92fbd42-ea2c-4843-85cd-653beff5c427","added_by":"auto","created_at":"2024-04-08 15:51:32","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":457128,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialTorabietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4146187/v1/cebf86bc6fc1b84115d70547.docx"}],"financialInterests":"Competing interest reported. SM is the CEO of miRas Biotech. miRas Biotech had no role in designing or analyzing the experiments. Other authors have no competing interests.","formattedTitle":"The RNAi enhancer enoxacin inhibits the growth and migration of esophageal squamous cell carcinoma cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEsophageal cancer is one of the deadliest and most aggressive types of cancer worldwide\u003csup\u003e1\u003c/sup\u003e. It has an extremely poor prognosis, and a 5-year survival rate of 15\u0026ndash;25%\u003csup\u003e2\u003c/sup\u003e. There are two main histopathological types of esophageal cancer including esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC) with the latter being the most prevalent form of esophageal cancer worldwide, accounting for more than 80% of all cases in some Asian countries such as China and Iran\u003csup\u003e3\u003c/sup\u003e. Although the currently available therapeutic approaches (i.e. curative surgery, chemotherapy, and radiotherapy) to ESCC have improved, the majority of these treatment strategies are not sufficiently effective, resulting in a marginal improvement in survival rates over time. Therefore, new therapeutic tools are needed to enable more effective treatment of ESCC patients\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicroRNAs (miRNAs) are a class of small, non-coding RNAs that post-transcriptionally regulate gene expression\u003csup\u003e5\u003c/sup\u003e. There have been numerous reports on the occurrence of mutations and disruptions in proteins, such as DICER, DROSHA, and TARBP2, which are directly involved in the biogenesis pathway of miRNAs in a variety of human cancers, including esophageal cancer. Such deregulations result in a global reduction in the expression of virtually all cellular miRNAs\u003csup\u003e6\u003c/sup\u003e. Alterations in the cellular miRNAome often cause major changes in the cellular proteome and consequently in the cell behavior\u003csup\u003e7,8\u003c/sup\u003e. Notably, global reductions in miRNA expression resulting from deregulations in miRNA biogenesis genes have been frequently observed to be associated with development and enhanced malignancy of several types of cancer including esophageal cancer\u003csup\u003e9,10\u003c/sup\u003e. Hence, stimulating the miRNA biogenesis pathway in esophageal cancer might be a viable option for treating the development and progression of this less-survivable disease.\u003c/p\u003e \u003cp\u003eThe fluoroquinolone family of antibiotics has been demonstrated to suppress the growth and invasiveness of multiple types of tumor cells without causing notable adverse effects on normal cells\u003csup\u003e11\u003c/sup\u003e. As shown in Table\u0026nbsp;1, in vitro and in vivo investigations on various types of cancer using several members of this family of antibiotics have shown a significant inhibitory effect on cancer cell growth and proliferation. Among all fluoroquinolones tested, enoxacin has frequently been found to have the most potent inhibitory effect on cancer cells\u003csup\u003e12,13\u003c/sup\u003e. It was revealed that the anti-cancer effects of enoxacin is exerted through the general enhancement of the RNA interference (RNAi) pathway, thereby stimulating the miRNA biogenesis pathway\u003csup\u003e14\u003c/sup\u003e. Enoxacin was then discovered to directly bind to the DICER\u0026rsquo;s physical partner TARBP2 and stimulate its protein activity leading to a general increase in the expression of cellular miRNAs which could bring the pattern of miRNA expression in cancer cells closer to that in the normal cells\u003csup\u003e15,16\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on these findings, we hypothesized that the RNAi enhancer enoxacin might be able to inhibit the viability and other growth features of ESCC cells in vitro. Our analysis revealed that enoxacin reproducibly suppressed the survival of ESCC cells compared to the control groups. It also significantly reduced the cell cycling as well as colony formation ability of the cells. Moreover, the migration of the ESCC cells was clearly reduced upon treatment with enoxacin. Finally, we found that enoxacin modulated the expression of several cancer-associated miRNAs through the simulation of the TARBP2 protein. Mechanistically, antisense oligonucleotides (ASOs) targeting \u003cem\u003eTARBP2\u003c/em\u003e transcript neutralized the inhibitory effects of enoxacin on ESCC cells, indicating that enoxacin exerts its anti-tumor effects on ESCC cells through stimulating \u003cem\u003eTARBP2\u003c/em\u003e activity. Overall, we report that globally enhancing the RNAi pathway leaves a potent inhibitory impact on ESCC cells.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eESCC cell lines (KYSE-30 and YM-1) and human foreskin fibroblast (HFF) cells were cultured in respective basal media, i.e. RPMI 1640 (Gibco) for KYSE-30, DMEM F12 (Gibco) for YM-1, or high-glucose DMEM (Gibco) for HFF cells, supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Invitrogen), 0.1% mM β-mercaptoethanol (Sigma-Aldrich), 1% Glutamax (Invitrogen), and 1% non-essential amino acids (Invitrogen). KYSE-30 cell line was a gift from Dr. Seyed-Javad Mowla lab at Tarbiat Modares University, Tehran, Iran. YM-1 cell line was a gift from Dr. Jahanbakhsh Asadi lab at Golestan University of Medical Sciences, Gorgan, Iran. Cells were maintained at 37\u0026deg;C in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e and cultured in T-25 vessels and other tissue-culture dishes (TPP). All cell lines were regularly tested for mycoplasma contamination using Hoechst staining and specific PCR Mycoplasma Detection kits. The media of the cells were changed every other day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEnoxacin treatment\u003c/h2\u003e \u003cp\u003eEnoxacin (Sigma-Aldrich) saline solution was prepared in water and stored at 4\u0026deg;C until further use. Enoxacin treatment was performed by adding the saline solution with a concentration of 124 \u0026micro;M to the culture medium. Due to the solubility of the salt form of enoxacin in water, no special substance or solvent was added to the culture medium in the control groups. Enoxacin has a limited water solubility and must be properly pipetted before use.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTransfection of\u003c/b\u003e \u003cb\u003eTARBP2\u003c/b\u003e \u003cb\u003eantisense oligonucleotides\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe oligonucleotides used in this study were synthesized by miRas Biotech (Tehran, Iran). \u003cem\u003eTARBP2\u003c/em\u003e-specific ASOs and scrambled (Scr) control oligonucleotides were delivered into the cells at a final concentration of 100 nM using DharmaFECT1 reagent (Dharmacon). The cells were cultured in culture dishes 24 h prior to transfection. Before doing transfections, the oligonucleotides as well as the DharmaFECT1 reagent were separately diluted in serum-free DMEM/F-12 media, the diluted oligonucleotides and the reagent were mixed at a 1:1 ratio, and then the resulting complex was kept at room temperature for 20 min. After oligonucleotide transfection, the cells were maintained in a 5% CO2 humidified incubator. The experiments were carried out in triplicate and the data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. 18 h post-transfection, the media of the cells were refreshed to remove the transfection reagent. Cells were analyzed 5 days post-treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eViability assays\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eMTS assay\u003c/h2\u003e \u003cp\u003eCell viability was evaluated using the MTS assay. In brief, 2.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells were seeded in 96-well cell culture plates and allowed to adhere overnight. Cell viability was measured 3 days post-treatment. To measure cell viability, the MTS solution was added to cell media in a 1:10 dilution ratio. The cells were then incubated at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e until a significant color change was observed (typically within 3 h). Finally, cell viability was measured by determining the absorbance at 495 nm using a microplate reader (ThermoFisher Scientific). The results were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026le;\u0026thinsp;0.05 in 3 biological replicates.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLive-dead assay\u003c/h2\u003e \u003cp\u003eTo perform live-dead assay, the cell supernatant was removed 3 days post-treatment, the cells were washed with PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e, and the dye solution of the live-dead assay was added to the cells. The dye solution, which is stored at 4\u0026deg;C and in dark before use, consists of calcine methyl ester (Calcein AM) and ethidium homodimer in a ratio of 1:5 in PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e. Cells were incubated in the presence of the dye solution for 20 min at 37\u0026deg;C, and were then viewed and imaged using fluorescence microscopy. In this assay, the green cells are live, the red cells are dead, and the yellow cells are dying cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCrystal violet staining\u003c/h2\u003e \u003cp\u003eThe cells were fixed with 4% paraformaldehyde solution at room temperature for 20 min, then washed with PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e. The crystal violet dye solution was diluted 1:50 in PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e and placed at room temperature. After 5 min, the cells were washed with PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e and visualized under a phase-contrast microscope. Crystal violet dye solution is prepared by dissolving 5 mg of the dye powder in 1 ml of 20% ethanol solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis\u003c/h2\u003e \u003cp\u003eThree days following treatment, 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were transferred to flow cytometry tubes and pelleted using centrifugation (5 min, 15000 rpm). After washing cell pellets 3 times with PBS, the nuclear DNA was fixed by adding 70% ice-cold ethanol is a dropwise manner along with low-speed vortexing followed by the incubation of the cells at 4\u0026deg;C for 2 h. Then, the cell pellet was washed twice with PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e to remove ethanol. In order to deactivate RNAs, 100 \u0026micro;l of RNase I was added to the cell pellet and incubated at 37\u0026deg;C for 20 min. Then, the cells were placed ice to inactivate the RNase enzyme. Cell cycle profiles were determined using flow cytometry with a FACSCalibur II instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eInvestigation of apoptosis by flow cytometry\u003c/h2\u003e \u003cp\u003eCells and their supernatant were collected separately 3 days post-treatment. The cells were then subjected to annexin V (Santa Cruz) and propidium iodide (PI, Sigma-Aldrich) staining using FITC Annexin V Apoptosis Detection Kit 1 (BD Biosciences). The rate of cell apoptosis was measured within 30 min post-staining using flow cytometry with a FACSCalibur II instrument. The data of early and late apoptosis and necrotic cells were analyzed by FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eTo analyze the colony formation ability of the cells, the cells were treated with enoxacin for 3 days, respectively. On day 3, the cells were trypsinized of which 200 cells were seeded into 35-mm dishes. The cells were then allowed to grow for 3 weeks to form colonies. Next, the colonies were stained with the crystal-violet dye solution, and the type and number of colonies were examined under a phase-contrast microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eTo investigate potential changes in the migratory behavior of the cells upon treatment, 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were cultured in each well of 12-well plates and treated for 3 days after reaching to ~\u0026thinsp;90% cell density. To attribute any changes in the area of the scratch to cell migration only, cell proliferation was inhibited by 1% mitomycin treatment for 18 h. Then, a scratch was made on the cells with the help of a yellow pipette tip under a loop microscope and the cells were then washed several times with PBS\u003csup\u003e\u0026minus;\u003c/sup\u003e. Images were taken every 24 h for 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative reverse-transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA extraction was performed using Qiagen\u0026rsquo;s miRNeasy mini kit, and RNA concentration and purity were measured using a spectrophotometer. The cDNA was synthesized using Thermo Scientific\u0026trade; RevertAid RT Reverse Transcription Kit for mRNA and Exiqon\u0026rsquo;s miRCURY LNA RT Kit for miRNA. The resulting single-stranded cDNA was stored at -80\u0026deg; C for long-term storage. PCR quantification of expression levels of gene transcripts was carried out using high ROX Takara Bio Green Premix Ex Taq II. The expression levels were normalized against the expression level of the endogenous control gene transcript \u003cem\u003eGAPDH\u003c/em\u003e. qRT-PCR analysis of miRNA was performed using SYBR green-based miRCURY LNA\u0026trade; Universal RT microRNA PCR system and LNA enhanced miRNA specific primers (Exiqon) according to the manufacturer\u0026rsquo;s instructions. miRNA expression levels were normalized against the small RNA endogenous control U\u003csub\u003e6\u003c/sub\u003e snRNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe results obtained in this study were analyzed using \u003cem\u003et\u003c/em\u003e test and one-way or two-way ANOVA depending on the number of variables by GraphPad Prism 9.0.0 software. Experiments were performed with at least 3 biological and 2 technical replicates. The data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and the significance of the data was represented as *\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEnoxacin has a cancer-specific growth-inhibitory effect on ESCC cells\u003c/h2\u003e \u003cp\u003eTo analyze the effects of enoxacin on ESCC cells, we first asked if enoxacin could reduce ESCC cell viability. The initial step was to determine the optimized enoxacin concentration to treat the cells. To this end, we cultured ESCC cells in 96-well plates, treated them for 5 days with different concentrations of enoxacin (0, 10, 25, 50, 75, 100, 125, 150, 200, 250, 300, and 350 \u0026micro;M), and measured ESCC cell viability using MTS assays. We observed that enoxacin dose-dependently suppressed the growth of ESCC cells, and determined its median inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) to be 125 \u0026micro;M (Supplementary Fig.\u0026nbsp;1a). Since several other studies have determined the IC\u003csub\u003e50\u003c/sub\u003e of enoxacin to be 124 \u0026micro;M\u003csup\u003e15,17\u003c/sup\u003e, we decided to perform all our subsequent treatments with 124 \u0026micro;M which is almost identical to the IC\u003csub\u003e50\u003c/sub\u003e that we determined in the current study. Notably, this inhibitory effect was observed to be cancer-specific, as enoxacin did not adversely affect the growth of non-cancerous cells i.e. human foreskin fibroblasts (HFFs), judging from crystal violet staining, MTS viability assessments, and live/dead staining (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb-d). We subsequently treated the ESCC cells with 124 \u0026micro;M of enoxacin continuously for 5 days for various experiments, as the 5-day treatment duration exerted a more potent inhibition than 3- or 4-day treatments on cell viability (Supplementary Fig.\u0026nbsp;1e). We also noticed that it was not necessary to treat the cells daily with fresh enoxacin as there was not a significant difference in cell viability when changing the media either every 24 h or every 48 h (Supplementary Fig.\u0026nbsp;1f). Therefore, we exposed the ESCC cells to enoxacin for 5 continuous days and refreshed the media of the cells every 48 h for the subsequent assays.\u003c/p\u003e \u003cp\u003eNext, the effect of enoxacin on growth and survival of the ESCC cell line, KYSE-30, was investigated. The cells were exposed to enoxacin for 5 days, and their survival was measured on day 5. We found that the number and viability of KYSE-30 cells were considerably decreased by enoxacin treatment compared to the untreated group, as evidenced by phase-contrast imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), crystal violet staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), and live/dead assays where enoxacin decreased the number of live (green) cells and simultaneously increased the number of dead (red) and dying (yellow) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d). This result was further confirmed using MTS viability assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) and the analysis of cell growth rate on the basis of cell counting following enoxacin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). We next repeated the same experiments in a second ESCC cell line, YM-1, to examine how reproducible the growth-inhibitory effects of enoxacin are. We obtained a highly-similar set of results for YM-1 cells and indicated that enoxacin potently inhibited the growth of YM-1 cells judging from phase-contrast imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), crystal violet staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), live/dead staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d), MTS viability assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), and cell counting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). These results indicate that enoxacin reproducibly inhibits the survival of ESCC cells whereas it has no inhibitory effects on the viability of normal cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eESCC cells undergo cell cycle arrest and apoptosis upon treatment with enoxacin\u003c/h2\u003e \u003cp\u003eAfter noticing a clear decline in survival of ESCC cells while being exposed to enoxacin, we looked at the cell cycle and apoptosis rate of the cells to examine if these two phenomena contribute to the decreased cell number observed upon enoxacin treatment. To determine the cell cycle profile, ESCC cells were seeded in 35-mm culture dishes, and were synchronized in G1 phase by being maintained under FBS-free conditions before the treatment. We found that 5 days treatment with enoxacin resulted in cell cycle arrest in the S phase of the KYSE-30 cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b) and in the G1 phase of YM-1 cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d), indicating that enoxacin impairs the cell cycling of the cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we sought to determine the apoptotic rate of the cells following exposure to enoxacin. Four days post-treatment, the KYSE-30 cells in the treatment and control groups were harvested and prepared for annexin-PI staining. Flow cytometry analysis of the cells stained with annexin-PI revealed that in the enoxacin-treated cells compared to the control group, (i) the number of live (non-apoptotic) cells was lower and (ii) the percentage of cells in the late phase of apoptosis was significantly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f). We also observed a similar increase in apoptosis rate when exposing YM-1 cells to enoxacin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h). Overall, enoxacin treatment induces cell cycle arrest as well as apoptosis in ESCC cells, thereby explaining the observed reductions in the number of ESCC cells upon treatment with the chemical.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEnoxacin inhibits capacity of ESCC cells to form colonies and migrate\u003c/h2\u003e \u003cp\u003eNext, we sought to determine whether enoxacin affects the clonogenicity of ESCC cells. To this end, the cells were treated with enoxacin for 5 days, and on day 5, the cells in both control and treatment groups were trypsinized, from which 200 cells were transferred to 35-mm cell culture dishes containing complete ESCC medium for colony formation. Within 3 weeks, several colonies emerged from the seeded cells which were then stained on day 21 with crystal violet solution and counted. The results indicated that enoxacin efficiently inhibited colony formation of KYSE-30 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The number and type of colonies were then analyzed and it was revealed that the colonies of the control group cells were mostly holoclones with a few colonies being meroclones or paraclones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c). In contrast, the colonies derived from the enoxacin-treated cells were more similar to cell clusters than cell colonies with few of them being paraclones (i.e. no holoclones and meroclones observed) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c). We also studied the YM-1 cell line in terms of clonogenicity in response to enoxacin exposure, and found a clear reduction in the number of colonies formed in the treated group compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f), confirming that enoxacin is reproducibly detrimental to colony formation by ESCC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we asked if enoxacin inhibits the ability of cancer cells to migrate. Toward this goal, we carried out wound healing (i.e. scratch) assays post-treatment and examined the scratch area over 24 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b, enoxacin considerably inhibited the closure of scratch wounds of the cultured cancer cells compared to the untreated cells. Since cancer cell migration is known to be dependent on cancer stem cells, we then analyzed the expression of several stemness genes e.g. \u003cem\u003eOCT4\u003c/em\u003e, \u003cem\u003eSOX2\u003c/em\u003e, \u003cem\u003eNANOG\u003c/em\u003e, \u003cem\u003eKLF4\u003c/em\u003e, \u003cem\u003ec-MYC\u003c/em\u003e, and \u003cem\u003eLIN28\u003c/em\u003e upon treatment with enoxacin. Our qRT-PCR analysis revealed that enoxacin suppressed the expression of almost all these genes 48 h post-treatment compared to the untreated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Moreover, it significantly decreased the expression of typical cancer-associated genes \u003cem\u003eALDH1\u003c/em\u003e, \u003cem\u003eCD44\u003c/em\u003e, and \u003cem\u003eCD133\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In conclusion, enoxacin blocks ESCC cell migration by downregulating key stemness and cancer genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTARBP2 mediates the anti-tumor effects of enoxacin in ESCC cells\u003c/h2\u003e \u003cp\u003ePrevious research has demonstrated that enoxacin induces cancer cell suppression by speeding up the maturation of miRNAs through binding and stimulating the TARBP2 protein, which is the physical partner of DICER1. To examine if enoxacin similarly promotes TARBP2-mediated processing of cellular miRNAs in ESCC cells, we treated the cancer cells with enoxacin and analyzed the expression of a selected set of miRNAs 48 h after exposure. It was observed using qRT-PCR assays that all the tested miRNAs were upregulated in response to enoxacin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), which was consistent with what reported previously\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we attempted to confirm whether TARBP2 is the mediator of enoxacin actions in ESCC cells. To this end, we first transfected the cells with a FITC-conjugated oligonucleotide to determine the efficiency of our transfection-based approach for the delivery of the \u003cem\u003eTARBP2\u003c/em\u003e-targeting ASOs. We observed that our lipid-based transient transfection method delivers the FITC-labelled oligonucleotides to the cultured ESCC cells in a highly efficient manner (~\u0026thinsp;70% of the cells received the oligonucleotide), as evidenced by fluorescence microscopy and flow cytometry (Supplementary Fig.\u0026nbsp;2a-b). Next, we suppressed the expression of \u003cem\u003eTARBP2\u003c/em\u003e transcript using a pool of two different \u003cem\u003eTARBP2\u003c/em\u003e-targeting ASOs with two different concentrations to determine the optimal ASO concentration for \u003cem\u003eTARBP2\u003c/em\u003e silencing, and found that a significant downregulation of \u003cem\u003eTARBP2\u003c/em\u003e transcripts could be similarly achieved upon treatment with either of the ASO concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), therefore we chose the lower concentration (100 nM) for transfection. Then, we treated the ESCC cells according to the procedure shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec to examine if \u003cem\u003eTARBP2\u003c/em\u003e knockdown abolishes the growth-inhibitory effects of enoxacin. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed-i, (i) enoxacin alone sharply reduced cell viability; (ii) \u003cem\u003eTARBP2\u003c/em\u003e-targeting ASOs had no inhibitory effects on ESCC cell growth; and (iii) enoxacin-ASO combination group did not reduce cell viability as potent as enoxacin alone, judging from phase-contrast imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed), crystal violet staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee), live/dead staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef-g), cell counting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eh), and MTS viability assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). Taken together, these results confirm that enoxacin suppresses the growth of ESCC cells by \u003cem\u003eTARBP2\u003c/em\u003e-mediated enhancement of miRNA maturation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer is the second leading cause of death worldwide. Esophageal cancer is one of the deadliest malignancies and less survivable cancers which is due to sub-optimal diagnostic tools and therapeutic strategies for the disease\u003csup\u003e18\u003c/sup\u003e. Iran and East Asia along with some Western societies have exceptionally high rates of this cancer type\u003csup\u003e19\u003c/sup\u003e. The majority of human cancers are known to have inactivating mutations in key components of the miRNA biogenesis pathway such as DROSHA, TARBP2, and DICER1, and exhibit a global downregulation in mature miRNA levels\u003csup\u003e20\u003c/sup\u003e. This suggests that not only cells\u0026rsquo; miRNAomes generally play a crucially important anti-cancer role\u003csup\u003e8,21\u0026ndash;23\u003c/sup\u003e, but also restoring a normal cell-like miRNA profile to cancer cells may be therapeutically-viable approach\u003csup\u003e8,24\u003c/sup\u003e. Therefore, agents that are able to globally enhance the RNAi (siRNA/miRNA biogenesis) pathway may serve anti-cancer functions.\u003c/p\u003e \u003cp\u003eEnoxacin is a member of the fluoroquinolone family of antibiotics that are frequently used to treat bacterial infections of the urogenital tract. In 2008, enoxacin was found to also influence the human (eukaryotic) cells by binding to TARBP2, the physical partner of DICER1 and potently enhancing the RNAi pathway\u003csup\u003e14,25,26\u003c/sup\u003e. It was later found that enoxacin\u0026rsquo;s stimulatory effect on the miRNA pathway promotes apoptosis in several types of cancer cells\u003csup\u003e15,27,28\u003c/sup\u003e. Since miRNAs play important roles in esophageal cancer cells\u003csup\u003e29\u0026ndash;31\u003c/sup\u003e, we hypothesized that enoxacin might inhibit these cells via enhancing the RNAi pathway. We observed that enoxacin treatment markedly reduced cell survival, ability to proliferation, colony formation, and migration ability of ESCC cells, but had no negative effects on the viability of normal cells as expected, because the drug had previously been approved by several international food and drug administrations to be safe for use in humans as an antibiotic. Notably, certain fluoroquinolones such as ciprofloxacin are currently used in the cancer treatment protocols to prevent or reduce the risk of wound infection following tumor tissue surgery\u003csup\u003e32\u0026ndash;34\u003c/sup\u003e, and in this sense, enoxacin may play a dual role and simultaneously serve as an anti-cancer and inti-infection drug for cancer patients.\u003c/p\u003e \u003cp\u003eMechanistically, we showed that enoxacin upregulated several randomly chosen miRNAs that already show expression in ESCC cells. To further corroborate the mechanism of enoxacin action, we silenced its major target protein TARBP2 using specific ASOs and found that enoxacin no longer inhibited the growth of ESCC cells upon TARBP2 knockdown. This result confirmed that enoxacin acts through TARBP2 to drive ESCC cell death. It would be worthwhile to analyze whether enoxacin could similarly inhibit the growth of ESCC cells in animal models of esophageal cancer.\u003c/p\u003e \u003cp\u003eIn summary, we showed that enoxacin considerably reduced the survival, cell cycling, and motility of esophageal cancer cells (KYSE-30 and YM-1 cell lines). Several key molecular players involved in the maintenance and progression of cancer stemness and malignancy of ESCC were observed to be suppressed by enoxacin. Moreover, we validated that enoxacin exerts its anti-tumor effects by stimulating TARBP2, thereby upregulating several tumor suppressing miRNAs. It is of critical importance to investigate the effect of enoxacin alone as well as in combination with other anti-cancer agents in animal models of ESCC (in vivo). Importantly, fluoroquinolones including enoxacin are small-molecule chemicals that can be taken orally and are known to exhibit a high bioavailability upon consumption by the patients. Although enoxacin is a small molecule that efficiently diffuses into the various tissues of the body, it may still be necessary to analyze the accessibility of the tumor cells to this drug. This is because under hypoxic conditions in the core of tumors, most of the cells located in this area are cancer stem cells and receive smaller concentrations of the drugs due to their less-developed vasculature. Cancer stem cells are resistant to various treatment regimens, therefore it would be important to investigate the exposure of ESCC cancer stem cells to enoxacin and examine how the stem cells respond. Further research would be required to reveal if enoxacin would have the potential to be used in humans as an anti-cancer drug.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank members of Moradi lab for critical discussions. This work was supported by a grant from Iran National Science Foundation (INSF) to Sharif Moradi (grant No. 98013813).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePT and HT performed the experiments, analyzed the data, and contributed to drafting of the manuscript. SRM served as scientific adviser. SM conceptualized, supervised, provided financial support, and wrote and approved the manuscript for submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available in the article and the associated supplementary file.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSM is the CEO of miRas Biotech. miRas Biotech had no role in designing or analyzing the experiments. 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A decrease in the global expression of microRNAs (miRNAs) is observed in various types of cancer, including esophageal cancer. It has been found that the small molecule enoxacin serves as an RNA interference (RNAi) enhancer, increasing the maturation rate of various cellular miRNAs. Here, we show that enoxacin significantly reduces the growth characteristics of ESCC cell lines. It induces cell cycle arrest and apoptosis in ESCC cells, leading to a clear decrease in ESCC cell number and viability. In addition, enoxacin suppresses the ability of cells to migrate and decreases their capacity to form colonies. Mechanistically, we reveal that enoxacin promotes the maturation of miRNAs through the stimulation of TARBP2 protein, the physical partner of DICER1. Taken together, enoxacin potently blocks the growth, motility, and clonogenicity of ESCC cells, paving the way for further investigation of this small-molecule chemical in animal models of ESCC.\u003c/p\u003e","manuscriptTitle":"The RNAi enhancer enoxacin inhibits the growth and migration of esophageal squamous cell carcinoma cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-08 15:43:26","doi":"10.21203/rs.3.rs-4146187/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8baf3ab6-d369-4828-b5bb-de492f701006","owner":[],"postedDate":"April 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30372904,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":30372905,"name":"Biological sciences/Cancer/Gastrointestinal cancer"},{"id":30372906,"name":"Biological sciences/Biotechnology/Oligo delivery"},{"id":30372907,"name":"Biological sciences/Biological techniques/Gene expression analysis"},{"id":30372908,"name":"Biological sciences/Biological techniques/Molecular engineering"}],"tags":[],"updatedAt":"2024-07-02T11:29:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-08 15:43:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4146187","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4146187","identity":"rs-4146187","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}