Cannabis sativa L. plant isolate, cannabidiol (CBD), has anti-cancer effects, which inhibit tumor growth and spread. This study investigated the cytotoxic combinative effect of PDT and CBD on CC HeLa cells. The effects were assessed by exposing in vitro HeLa CC-cultured cells to varying doses of ZnPcS4 photosensitizer (PS) PDT and CBD, with a fluency of 10 J/cm2 and 673 nm irradiation. HeLa CC cells, which received the predetermined lowest dose concentrations (ICD50) of 0.125 µM ZnPcS4 PS plus 0.5 µM CBD to yield 50% cytotoxicity post-laser irradiation, reported highly significant and advantageous forms of cell death. Flow cytometry cell death pathway quantitative analysis showed that only 13% of HeLa cells were found to be viable, 7% were in early apoptosis and 64% were in late favorable forms of apoptotic cell death, with a minor 16% of necrosis post-PDT. Findings suggest that this combined treatment approach can possibly induce primary cellular destruction, as well as limit CC metastatic spread, and so warrants further investigation.The most prevalent type of gynecological malignancy globally is cervical cancer (CC). Complicated by tumor resistance and metastasis, it remains the leading cause of cancer deaths in women in South Africa. Early CC is managed by hysterectomy, chemotherapy, radiation, and more recently, immunotherapy. Although these treatments provide clinical benefits, many patients experience adverse effects and secondary CC spread. To minimize this, novel and innovative treatment methods need to be investigated. Photodynamic therapy (PDT) is an advantageous treatment modality that is non-invasive, with limited side effects. The  Cervical cancer (CC) is the fourth most frequently detected cancer in females and presents a considerable health burden to women globally , warrant1O2). The generation of these cytotoxic species induces oxidative stress, which can cause primary CC cell death via apoptosis [Photodynamic therapy (PDT) is an alternative treatment modality, which has presented support of CC primary abolition . The mecpoptosis . The antpoptosis .2+ in the Pc nucleus, seems to improve the triplet state lifetime, as well as its yield and singlet oxygen yields compared to paramagnetic metals, however, the metalation is not required for its photodynamic activity [Cancer research into the prospective use of phthalocyanines (Pcs) as PSs in PDT is favorable. This is attributed to their superior tumor passive uptake, high ROS production, and potent absorption in the 680 nm red wavelength of light . Moreoveactivity .A review by Carobeli et al. (2021) indicates that Pc PSs have potential as PDT pharmaceutical agents for anti-CC therapy. The authors firmly believe that Pc-based PS formulations could allow for the development of lead PDT compounds for the primary treatment of CC . Howevern PSs have received particular attention as PDT agents over the years, since sulfonation of the substituted ZnPc enhances its solubility to aggregate less and promote its uptake in tumor tissues, with improved in vitro and in vivo PDT outcomes [The potential of sulfonated Pcs for clinical PDT has prompted a search for alternate synthetic approaches to yield well-characterized compounds as single isomeric products . Hydrophoutcomes . Howeveroutcomes ,14,15. 4) has been designed as an amphiphilic PS agent to promote uptake, as well as circumvent any issues associated with solubility and aggregation [4 PSs, can efficiently bind to cancer tumor phospholipid membranes, through metal–phosphate coordination [4 PSs can spontaneously accumulate in tumors through an enhanced permeability and retention (EPR) effect, due to the binding of its tetrasulfonated groups to cancer cell membranes (which have high phospholipid contents), and so allow for passively selective accumulation [Zinc phthalocyanine tetrasulfonate , was an effective PS within in-vitro-cultured HeLa cells, when treated with 4 J/cm2 at a wavelength of 673 nm, producing 50% cytotoxicity [2) PS within in vitro HeLa cells, and noted that the PS was capable of subcellular localization and that this PDT inhibited mitochondrial respiration in hypoxic conditions [4 PS within in vivo human normal versus cancerous colon tissue samples, and demonstrated that this PS had a lower affinity for normal tissues [4 PS had a monomer region signal, confirming its ideal PS properties within PDT applications [4 PS were capable of endoplasmic reticulum (ER) localization, with mid concentrations capable of mitochondrial and/or Golgi apparatus lysosome localizations, while reporting preferential localization in the nucleus of colon cancer tissues at higher concentrations [4 PS in MCF-7 breast cancer cells, with significant cytotoxic effects reported [Studies by Hodgkinson et al. 2017) demonstrated that sulphonated ZnPc, containing a mixture of differently sulfonated derivatives  (PLGA) NPs and research by Naidoo et al. (2019) showed excellent active targeting subcellular uptake, with enhanced PDT treatment outcomes when conjugating ZnPcS4 PSs and an antibody PLGA gold NP delivery carrier, within in vitro melanoma cancer cultured cells, suggesting that both carriers could potentially serve as bioactive models [4 PS PDT treatment outcomes within in-vitro-cultured colorectal cancer cells, when conjugating to an antibody actively targeted PLGA-loaded gold NP or loading it in core/shell Ag@mSiO2 NP with folic acid, respectively [4 PSs, was developed. Results reported excellent PDT anti-tumor activity within in vivo Swiss albino mice, using an Ehrlich solid tumor as an experimental model for breast cancer [4-AN was capable of mediating PDT to refrain tumor aggressiveness, as well as induce regression [4-AN-mediating PDT exposed anti-neoplastic activity, such as that obtained while using intra-tumoral conventional chemo-Dox therapy [4 PSs in vivo interstitially, to attempt to bring this platform closer to clinical investigations [4 PS did not elicit notable systemic toxicity in zebrafish and chicken embryos, and in human tumor breast cancer xenografts, it produced a significant tumor reduction. However, it did report skin phototoxicity in mouse models [According to Rak et al. (2019), other options to increase the bioavailability and solubility of metallo-phthalocyanine (MPc) PSs in cancer PDT to translate it into future in vivo and clinical trial research is by using various carrier delivery systems, such as nanoparticles (NPs), nanoemulsions, and liposomes, among others . De Tolde models ,24. Simiectively ,25. In at cancer . Intratugression . Moreove therapy . More reigations . The keye models . This ste models .4 PS within PDT has, thus, been extensively researched in the cancers mentioned above, and has presented some promising results; however, it has not been researched within in vitro or in vivo models for possible shortcomings in CC. It was therefore decided to investigate this PS within this novel study, to provide a platform for future clinical development. Researchers do note that there is still a lot of in vitro work to be done to improve ZnPcS4 PS PDT in relation to its penetrability to tumor tissues, selectivity, stimulating methods, as well as promote its overall ability to overcome tumor hypoxia microenvironment to translate its use into future in vivo models. The utilization of ZnPcS4 PS PDT real clinical translational potential, investigations in combination with other treatments are needed. A study by Shams and colleagues (2015) suggested a need to develop PDT regimens which eradicate primary tumor growth, as well as inhibit metastasis through host immune system stimulation [Dicoma anomala root extract in combination with ZnPcS4-PS-mediated PDT in A549 lung cancer cells. This combination therapy confirmed the cytotoxic and anti-proliferative effects of Dicoma anomala extracts in monotherapy and in combination with ZnPcS4-mediated PDT, through apoptosis and the upregulation of p38, p53, Bax, caspase 3, 8, and 9 apoptotic proteins, suggesting that combinative therapies are worth exploring [Moreover, even though research has reported that PDT has emerged as a possible, effective, and tolerable treatment approach for the management of primary CC, it also requires improvement in terms of investigating combinative PDT treatments to stimulate specific immune responses to eliminate secondary spread . Overallmulation . Chota exploring . Cannabis sativa L. plant [Cannabinoids are a group of naturally occurring metabolites located abundantly in the L. plant . ResearcL. plant . The effL. plant . The eviL. plant .4 PS carrier as a PDT treatment for in-vitro-cultured colorectal cancer in combination with CBD allows for targeted primary tumor destruction, as well as activation of specific immune responses, which limit metastasis [More recently, Nkune, Kruger, and Abrahamse (2022) also reported encouraging results, demonstrating that utilizing antibody-targeted PLGA-loaded gold NP ZnPcStastasis . Lukheletastasis . Therefo4 PS and CBD on the survival of in-vitro-cultured HeLa cells, to determine its potential application as a combinative complementary treatment form for CC. Since this is a novel study and the combinative effect of ZnPcS4 PS PDT and CBD has never been investigated within in vitro CC, the intention of this research was to lay the groundwork foundation in terms of investigating its potential effectiveness. These findings will possibly allow for future investigations in relation to enhancing the delivery of this combinative treatment with various drug targets and carriers, to promote its possible in vivo effectiveness. Thus, the aim of this study was to investigate the PDT-mediated effect of ZnPcS4 PS PDT on HeLa cells, experimental and control groups were treated with different concentrations of ZnPcS4 PS . After laser irradiation, culture plates were re-incubated for an additional 24 h prior to being subjected to lactate dehydrogenase (LDH) membrane damage integrity analysis. Cellular lysis was induced and 100% LDH release was noted in the positive control, whereas the cells-only control indicated 3% (±SEM 0.47) cytotoxicity, and were used for statistical comparisons. The control groups of HeLa CC cells, which received increasing concentrations of ZnPcS4 PS  alone, without any influence of irradiation, respectively, reported an insignificant incremental average increase (±16%) in cellular cytotoxicity  cytotoxicity, while for experimental groups which received 0.125 µM ZnPcS4 PS and PDT, 47%** (±SEM 1.25) cytotoxicity was somewhat significant. However, experimental groups which received 0.25 µM and 0.5 µM ZnPcS4 PS, as well as PDT, produced highly significant increases of 53%*** (±SEM 0.47) and 57%*** (±SEM 0.48) in their respective cytotoxicity assays. The most significant increase in cytotoxicity of 63%*** (±SEM 0.82) was reported in experimental groups, which received 1 µM ZnPcS4 PS and PDT. The experimental groups of HeLa cells which received increasing concentrations of ZnPcSv/v) ethanol only, generated an incremental increase of 14% (±SEM 0.34) in cellular cytotoxicity. The experimental group, which received 0.3 µM CBD and irradiation, reported 43%** (±SEM 1.05) cytotoxicity, which was somewhat significant. However, experimental groups which received 0.5 µM and 0.7 µM CBD, as well as irradiation, denoted significantly increased values of 52%** (±SEM 0.75) and 56%** (±SEM 0.78), with reference to their respective cytotoxicity assays. The most significant increases in cytotoxicity of 57%** (±SEM 0.86) and 62%*** (±SEM 0.42) were reported in the experimental groups which received 0.9 µM and 1.1 µM CBD plus irradiation, respectively. Similar overall average cytotoxicity results were reported for control groups consisting of cells plus CBD of ±53%** . The control groups of HeLa cells which received 99.8%  viable population of cells, with negligible cell death. The positive Annexin-V-FITC-stained control of apoptotic-induced cell death denoted only 12% (±SEM 0.45) of viable cells, a significant 49% (±SEM 0.48) of early apoptosis and 37% (±SEM 0.41) of late apoptosis, as well as a minor 2% (±SEM 0.51) of necrotic cell death. The negative PI-stained control of necrotic-induced cell death denoted only 10% (±SEM 0.57) of viable cells, a minor 8% (±SEM 0.61) of early apoptosis, and 6% (±SEM 0.56) of late apoptosis; however, a significant 76% (±SEM 0.49) necrotic cell death was also observed. Thus, these positive controls were considered as acceptable standards for result comparisons and statistical data interpretations. v/v) of ethanol only, reported a majority of 87% (±SEM 0.53) of viable cells, without any other significant forms of cell death. The control group of HeLa cells plus irradiation reported a substantial 92% (±SEM 0.92) of viable cells, without any other significant forms of cell death. The control group of HeLa cells, which received 99.8%  of viable cells, without any other significant forms of cell death. The HeLa cells which received 0.125 µM ZnPcS4 PS and irradiation reported a significant reduction in viability, whereby only 52%* (±SEM 0.87) of cells were noted as viable. Furthermore, 15% (±SEM 0.92) of these cells were undergoing early apoptosis, and an even more significant 24%** (±SEM 0.85) were in a late apoptotic form of cell death, with an incremental amount of 9% (±SEM 0.56) necrosis. The control group of HeLa cells which received 0.125 µM ZnPcSThe control and experimental groups of HeLa cells which received 0.5 µM CBD alone, with or without laser irradiation, respectively, reported similar significant forms of cell death. The control group which received 0.5 µM CBD without irradiation noted a significant decrease of only 46%** (±SEM 0.44) of cells being viable, as well as a significant increase of 38%** (±SEM 0.46) of cells undergoing late apoptosis, with incremental amounts of early apoptosis and necrosis. Similarly, the experimental group which received 0.5 µM CBD with irradiation noted a significant decrease of only 50%** (±SEM 0.56) of cells being viable, as well as a significant increase of 42%** (±SEM 0.62) of cells undergoing late apoptosis, with incremental amounts of early apoptosis and necrosis. 4 PS plus 0.5 µM CBD, without PDT, reported a significant reduction, whereby only 35%** (±SEM 0.76) of cells were viable. Furthermore, 11% (±SEM 0.66) of these cells were undergoing early apoptosis and an even more significant 45%** (±SEM 0.72) of these cells were in a late apoptotic form of cell death, with an incremental amount of 9% (±SEM 0.55) necrosis. The experimental groups of HeLa cells which received 0.125 µM ZnPcS4 PS plus 0.5 µM CBD and irradiation reported the most highly significant and favorable forms of cell death, with only 13%*** (±SEM 1.04) of the cells being viable. A further 7% (±SEM 0.98) of the cells were in early apoptosis and a confounding 64%*** (±SEM 0.93) were in late forms of favorable apoptotic cell death, with a minor 16% (±SEM 1.12) of necrosis post-PDT. In comparison to the experimental groups of HeLa cells which received 0.125 µM ZnPcS4 PS plus 0.5 µM CBD without PDT, the percentages of cellular viability were significantly decreased (35%** vs. 13%**) and the percentages of late apoptotic cell death were also significantly increased (45%** vs. 64%***). In contrast, HeLa cells which received 0.125 µM ZnPcS4 PS and CBD PDT/irradiation combinative assays, are shown. With reference to The cells-only positive control noted a 98% (±SEM 0.51) viable population of cells, with negligible cell death. The positive Annexin-V-FITC-stained control of apoptotic-induced cell death noted only 10% (±SEM 0.73) of viable cells, a significant 34% (±SEM 0.81) of early apoptosis and 52% (±SEM 0.79) of late apoptosis, as well as a minor 4% (±SEM 0.84) necrotic cell death. The negative PI-stained control of necrotic-induced cell death noted only 8% (±SEM 0.32) of viable cells, a minor 6% (±SEM 0.46) of early apoptosis and 8% (±SEM 0.39) of late apoptosis; however, a significant 78% (±SEM 0.41) necrotic cell death was also observed. v/v) of ethanol only, reported a majority of 88% (±SEM 1.01) of viable cells, without any other significant forms of cell death. The control group of WS1 cells plus irradiation reported a substantial 96% (±SEM 0.60) of viable cells, without any other significant forms of cell death. The control group of WS1 cells which received 99.8%  cells which received 0.125 µM ZnPcS4 PS and irradiation, did not report significant forms of cell death, however, in comparison to normal cells, only 74% (±SEM 0.63) remained viable, with an 8% (±SEM 0.67) increase in late apoptosis. The control group of WS1 cells which received 0.125 µM ZnPcSThe control and experimental groups of WS1 cells which received 0.5 µM CBD with or without laser irradiation, respectively, reported no significant forms of cell death. The control group which received 0.5 µM CBD without irradiation noted 87% (±SEM 0.58) of cells being viable, whereas the experimental group which received 0.5 µM CBD with irradiation similarly noted 86% (±SEM 0.87) of cells being viable. 4 PS plus 0.5 µM CBD without irradiation also reported no significant reduction in cell viability, with incremental amounts of cell death. Similarly, the same results were reported in control groups which received 0.125 µM ZnPcS4 PS alone or 0.5 µM CBD alone. The experimental groups of WS1 cells which received 0.125 µM ZnPcS4 PS plus 0.5 µM CBD with laser irradiation noted no significant forms of cell death and 73% (±SEM 0.93) of the cell population remained viable. However, in comparison to the cells-only control group, there was a higher population of early (10% ±SEM 1.03) and late apoptosis (13% ±SEM 0.99) being noted. Similar findings were reported in control groups which received 0.125 µM ZnPcS4 PS and PDT, whereby 12% (±SEM 0.77) of early and 8% (±SEM 0.47) of late apoptosis was noted. Subsequently, control groups of WS1 cells which received 0.5 µM CBD with irradiation, respectively, showed slightly less (7% ±SEM 0.86) early apoptosis and 4% (±SEM 0.79) of late apoptosis. The experimental group of WS1 cells which received 0.125 µM ZnPcSNonetheless, even though the cell death was insignificant, since minor early apoptosis/autophagy was observed, there was a possibility that WS1 post-PDT could recover, which required corroboration in ATP proliferation assays.4 PS and CBD PDT/irradiation combinative assays, has been shown. With reference to The cells-only positive control, which received no forms of treatment, noted a 100% (±SEM 1.07) viable population of cells capable of cellular proliferation, and was thus considered an acceptable standard for result comparisons and statistical data interpretations.v/v) of ethanol or 0.125 µM ZnPcS4 PS only, noted no significant decreases in cellular proliferation. However, the control groups of HeLa cells which received 0.125 µM ZnPcS4 PS and laser irradiation reported a significant decrease, as only 20%*** (±SEM 0.82) of the cell population post-PDT treatment was able to proliferate. The control groups of HeLa cells which received irradiation, 99.8%  and 23%*** (±SEM 0.82) of the cell populations pre- and post-PDT treatment, respectively, were capable of proliferation. 4 PS plus 0.5 µM CBD without PDT reported a significant reduction, whereby only 18%*** (±SEM 0.82) of the cells maintained their ability to proliferate, whereas HeLa cells which received 0.125 µM ZnPcS4 PS plus 0.5 µM CBD and PDT reported the most significant decreases in cellular proliferation, noting that only 6%*** (±SEM 0.94) of the cellular population was capable of proliferation. The experimental groups of HeLa cells which received 0.125 µM ZnPcS4 PS and CBD PDT/irradiation combinative assays, has been shown. With reference to The positive control of cells only noted a 100% (±SEM 0.98) viable population of cells, capable of cellular proliferation and so was considered as an acceptable standard for result comparisons and statistical data interpretations. 2, showed increases in cellular cytotoxicity (9%)  . This isectively ,34,36. T4 PS alone without any influence of irradiation, respectively, reported insignificant incremental average increases (±16%) in cellular cytotoxicity , remained inactive and lacked any form of dark phototoxicity. Second-generation ZnPc Pcs, such as ZnPcS4 PSs, have shown high stability in the absence of light [The control groups of HeLa cells which received increasing concentrations of ZnPcStoxicity . The outof light ,37,38,394 PS in these dose concentration ranges, could become excited and sufficiently activated to produce considerable amounts of ROS and 1O2 cytotoxic species. This also implies that it is capable of significant in-vitro-cultured HeLa primary cellular destruction. Related studies have stated that since ZnPcS4 is a metalated second-generation Pc PS, its cytotoxic PDT treatment outcomes for cancer are often highly favorable and it allows for activation within the far-red spectral range of 634 to 674 nm. This permits for maximum tissue depth penetration and excitation, and high yields of localized PDT-induced cytotoxic species, which in turn can cause considerable primary tumor destruction [Findings for this assay suggest that laser irradiation, with the administered ZnPcStruction ,37,38,3950 ZnPcS4 PS concentration able to induce approximately 50% cytotoxicity within in-vitro-cultured HeLa experimental groups, was found to be 0.125 µM. This concentration was chosen since it reported 47%** cytotoxicity, as it ensured that HeLa cells remained somewhat viable post-PDT, so that their overall combinative biological and cell death effects could be measured. The determined ICD4 PS PDT primary tumor destruction has not yet been investigated within in-vitro-cultured HeLa cells, these cytotoxicity results seem promising, as the ICD50 results at a concentration of 0.125 µM were much lower than in previous studies by Hodgkinson et al. (2017) at 1 μM of ZnPcSmix PS, and by Pola et al. (2021) at 0.5 μM ZnPcS2 PS [Since hydrophilic ZnPcSn et al. 17 at 1 μet al. 20 at 0.5 μ2 caused negligible cellular viability or cytotoxicity effects on their in-vitro-cultured HeLa controls [The control groups of HeLa cells which received laser irradiation showed slight increases in cellular cytotoxicity (10%) . This iscontrols .v/v) ethanol showed 14% cellular cytotoxicity, suggesting that the CBD solvent played no substantial role in the treatment outcomes. Supportively, studies by Ramer et al. (2010) which investigated the treatment effects of CBD on cultured HeLa cells reported that CBD solvents, such as ethanol, which were individually controlled for within assays, had no contributing cytotoxic effects on the treatment outcomes either [The control groups of HeLa cells which received 99.8%  in cellular cytotoxicity. These results showed that as the concentrations of CBD increased, cellular cytotoxicity proportionally increased. The determined ICD4 PS PDT on in-vitro-cultured colorectal cancer cells, whereby CBD individual dose response assays  were performed on control groups which received CBD alone and experimental groups which received CBD and laser irradiation [These significant findings mirrored the results observed within the experimental groups of HeLa cells which received the same linear increments of increasing concentrations of CBD with laser irradiation, when compared to their respective cells-only control groups. The results suggest that irradiation had no influence on the CBD treatment effects, and that the induced highly significant increases in cytotoxicity could be attributed to the therapeutic effects of CBD alone. Nkune et al. (2022) investigated the synergistic treatment effects of CBD and ZnPcSadiation . The outadiation . Moreoveadiation . This stadiation .4 PS, and experimental groups which received 0.125 µM ZnPcS4 PS + 0.5 µM CBD, the amount of ZnPcS4 PS uptake appeared fairly equal, and so it can be assumed that CBD had no effect upon the absorption of ZnPcS4 PS into the CC HeLa cells. This correlates with the study by Nkune et al. (2022), which investigated the combinative treatment effects of ZnPcS4 PS and CBD on colorectal cancer cells [4 PS, with or without the addition of CBD, remained the same, and concluded that its presence would not affect the subcellular localization of this PS [As can be observed from both the control groups of HeLa cells in er cells . They no this PS . 4 PS was able to enter the cells successfully and was possibly able to mainly sub-localize in the cytoplasm, rather than in the cellular membrane. Carobeli et al. (2021) also stated that metallo-Pc PS formulations, which exhibit higher capacities to permeate the cytoplasmic membrane and internalize within the cell cytoplasm, reported far more effective PDT outcomes [4 PS was able to permeate the cytoplasmic membrane of HeLa cells, it would have the opportunity to sub-localize within the mitochondria and lysosomes of HeLa cells, and so possibly be able to induce the most desired mechanism of programmed cell death in PDT, which is apoptosis and not necrosis [Furthermore, from both images taking the placement of the green-stained CC HeLa cell membrane and their blue-stained nucleus in overlay images into account, it can be observed that the ZnPcSoutcomes . This isoutcomes . Pazos aoutcomes . These qnecrosis . 4 PSs can efficiently bind to tumor phospholipid membranes, through metal–phosphate coordination and spontaneously accumulate in tumors through an EPR effect. This is attributed to its tetrasulfonated groups passively high affinity toward cancer cell membranes [4 PSs have a higher affinity for colon cancerous human tissues than normal tissues, as well that lower concentrations of ZnPcS4 PS were capable of ER localization [Necrosis is described as a rapid and relatively broad mechanism of cell death, characterized by vacuolization of the cytoplasm and cell membrane destruction. A localized inflammatory reaction is subsequently induced due to the release of cytoplasmic content and pro-inflammatory mediators in the extracellular space . High doembranes ,17,19. Ilization .4 PS and experimental groups which received 0.125 µM ZnPcS4 PS + 0.5 µM CBD, there was a slight uptake of the ZnPcS4 PS in the normal control cells, regardless of whether CBD was present. The subcellular localized uptake of PS is an essential factor in PDT, since it determines PDT potency and the PS’s direct passive or active intracellular localization at the tumor site, which in turn can enhance overall cell death treatment outcomes [4 PS red autofluorescence, when compared to the same control and experimental groups of HeLa cells in 2 PSs in HeLa cells versus normal human fibroblast lung WI-38 cells [2 PSs showed potential for CC PDT treatment due to their ability to localize within the cytoplasmic region of CC cells more passively compared to normal human cells [2 PSs did show slight absorption in the normal human control cells, when laser irradiation was applied to a localized tumor, some of the normal surrounding healthy tissues became compromised. Nevertheless, the benefits of such treatment outcomes to eradicate primary tumor cells would outweigh the negligible damage caused in normal tissues, since the laser activation of PS for effective treatment outcomes remains localized [4 PS remained unaffected in relation to subcellular localization in the presence of CBD. Additionally, since it was able to accumulate within in-vitro-cultured CC HeLa in a more concentrated manner, than when compared to normal WS1 fibroblast cells, these findings hint that it should be capable of more localized PDT treatment outcomes. Since the above images were captured utilizing fluorescent microscopy with fixed cells, there is a possibility that they could contain fixation-induced artifacts in the distribution of ZnPcS4 PS, which can affect their overall resolution, and so they can only be utilized for quantitative observations with hypothetical conclusions. Further confirmatory confocal microscopy studies with live cells need to be performed and measured to confirm the possible sub-cellular localization abilities of ZnPcS4 PS.As can be observed from both control groups of WS1 fibroblast cells in outcomes . However38 cells . They no38 cells . The resan cells . Howeverocalized . Overallmix PS [The control group of HeLa cells plus laser irradiation reported a substantial 92% of viable cells, without any other significant forms of cell death . Chizengmix PS .v/v) of ethanol only, reported 87% viable cells without any other significant forms of cell death, suggesting that this solvent played no substantial role within the combinative treatment outcomes. Ramer et al. (2010) also noted that CBD ethanol solvent alone within in-vitro-cultured HeLa cells had no contributing cytotoxic effects on the treatment outcomes [The control group of HeLa cells which received 99.8% . These results confirmed that the chosen ICD50 0.125 µM ZnPcS4 PS concentration was capable of red laser light excitation, as well as yielding sufficient ROS and 1O2 cytotoxic species to induce an overall 48% cell death . The control groups of HeLa cells which received 0.125 µM ZnPcSPDT can induce three types of cell death in cancer: type I (autophagy/early apoptosis), type II (late apoptosis), and type III (necrosis) . When PD4 PS and laser irradiation reported 15% of cells undergoing early apoptosis and a significant 24%** of cells in a late apoptotic form of cell death, with an incremental amount of 9% necrosis, it can be safely assumed that this treatment form was capable of primary CC cell death, with minimal inflammation. Moreover, these outcomes support the subcellular localization findings, whereby it was reported that 0.125 µM ZnPcS4 PS were significantly localized in HeLa cells, signifying that this PS was capable of subcellular localization and favorable apoptotic forms of cell death post-PDT. Since control groups of HeLa cells which received 0.125 µM ZnPcS4 PS and irradiation noted significant amounts apoptotic cell death post-PDT, these results are suggestive of the ZnPcS4 PS ability to permeate the cytoplasmic membrane of HeLa cells and sub-localize within the mitochondria and lysosomes. Metallo-Pc PS formulations which exhibit higher capacities to permeate the cytoplasmic membrane and internalize within the cell cytoplasm report far more effective PDT outcomes, and since ROS has a short half-life, when a PS becomes excited, the closer it is internalized within a cancer cells contents and nuclei, the far better the controlled intra-tumoral apoptotic destruction is [Since control groups of HeLa cells which received 0.125 µM ZnPcSction is ,42. Studction is ,41.50 0.5 µM CBD concentration was capable of cytotoxic cell death, since it induced an overall approximate 50% cell death (of which a significant 38%** pre-laser and 42% post-laser irradiation treatments were late apoptotic). The Lukhele and Motadi (2016) study attributed the significant increases of cell death to the various secondary metabolites in CBD, which stimulate various cellular signaling pathways to generate late apoptotic and anti-tumor proliferation responses in cancer cells [These results confirmed that the chosen ICDer cells . Comparaer cells ,40,47. Wer cells . 4 PS plus 0.5 µM CBD without PDT reported a significant reduction in cell viability (35%**), and an even more significant 45%** of these cells were in a late apoptotic form of cell death. The significant cell death findings in the above results were very similar and comparable to the control groups of HeLa cells which received 0.5 µM CBD without irradiation. Since the control groups of HeLa cells which received 0.125 µM ZnPcS4 PS without PDT noted no significant forms of cell death due to the PS remaining inactive, this significant cell death could only be accredited to the treatment effects of CBD.The experimental groups of HeLa cells which received 0.125 µM ZnPcS4 PS plus 0.5 µM CBD and PDT reported the most significant and favorable forms of cell death. Only 13%*** of the cells were viable and a significant 64%*** were in late forms of favorable apoptotic cell death. In comparison to the HeLa cells which received 0.125 µM ZnPcS4 PS plus 0.5 µM CBD without irradiation, the percentages of cellular viability were significantly decreased (35%** vs. 13%**) and the percentages of late apoptotic cell death were significantly increased (45%** vs. 64%***). Since the only difference between these two experimental groups was that irradiation was present, these findings suggested that the ZnPcS4 PS was successfully activated with laser irradiation and that this excitation was able to produce high yields of cytotoxic species, which promoted these substantial primary forms of late apoptotic cell death (64%***) in HeLa cells. The experimental groups of HeLa cells which received 0.125 µM ZnPcS2 did not affect this treatment pre-PDT. Furthermore, the application of laser light did not affect CBD’s ability to induce HeLa cell death. The ZnPcS4 PSs noted a high photochemical stability in the absence of light [1O2 that could induce significant forms of late apoptotic cell death [4 PSs were capable of significant amounts of programmed late apoptotic cell death, suggesting that this combinative treatment post-PDT would not induce any unwanted inflammatory responses or allow primary HeLa cells to recover [Overall, these combinative experimental assays concluded that laser parameters at a wavelength of 673 nm and fluence of 10 J/cmof light , and werll death . Both CB recover .4 PS. These findings are supported by studies performed by Cios et al. (2021), which stated that red laser applications within the wavelength of 620 to 740 nm, without any additions of PS components, noted no difference in WS1 in-vitro-cultured biologic responses [The control groups of cells reported viable population of cells, with negligible cell death . These fesponses .v/v) of ethanol only reported a majority of 88% of viable cells, without any other significant forms of cell death. Kar et al. (2021) stated that doses of ethanol lower than 10% (v/v) reported significant effects on in-vitro-cultured mouse fibroblasts cellular proliferation and viability; however, doses any higher than this, especially those utilized at clinical levels above 80% (v/v), remained non-toxic to the cells [The control group of WS1 cells which received 99.8% , which investigated the use of AlClPc as a possible photosensitizing agent for cancer PDT applications, noted no side effects in the normal in-vitro-cultured microenvironment of fibroblasts, in the absence of laser irradiation [The control group of WS1 cells which received 0.125 µM ZnPcSadiation .4 PS and PDT, did not report significant forms of cell death, suggesting that only slight cellular damage to normal human fibroblasts occurred. Insignificant early stages of apoptosis are often indicative of autophagy, whereby cells can sometimes recover [4 PS in the control normal cells, however it was far less when compared to the same control and experimental groups of HeLa cells. The control groups of WS1 cells which received 0.125 µM ZnPcS recover ,52. SimiThe control group of WS1 cells which received 0.5 µM CBD without irradiation noted 87% of cells being viable, whereas the experimental group which received 0.5 µM CBD with irradiation similarly noted 86% of cells being viable. Supportively, Pagano et al. (2020) reported that CBD doses ≤ 10 µM noted no cytotoxic effects on normal human healthy cell populations . 4 PS plus 0.5 µM CBD with PDT noted no significant forms of cell death. However, in comparison to the cells-only control group, there was a higher population of early (10%) and late apoptosis (13%) being noted. Similar findings were reported in control groups which received 0.125 µM ZnPcS4 PS and PDT. Subsequently, control groups of WS1 cells which received 0.5 µM CBD with irradiation, respectively, showed slightly less (7%) early apoptosis and 4% of late apoptosis. Similarly, Souza et al. (2021) and Pagano et al. (2020) noted that neither red laser irradiation nor CBD caused unwanted cytotoxic effects in WS1 cells, respectively [2 PSs showed a four-times higher intracellular uptake in the HeLa cells when compared to normal cell lines. The results concluded that even though there was slight absorption of the PS in normal cells, since laser irradiation was a localized tumor treatment, only some of the normal surrounding healthy tissues could become compromised [The experimental group of WS1 cells which received 0.125 µM ZnPcSectively ,53. Avşapromised . v/v) of ethanol solvent and the ZnPcS4 PS did not influence any combinative treatment outcomes in HeLa cells  was found. As discussed above, Mishchenko et al. (2022) reported that the most favorable form of PDT-induced cell death is late apoptosis; however, if cancer cells undergo early apoptosis post-PDT, some could be in an autophagy state, which can simultaneously induce pro-survival and unwanted recovery [4 PS and PDT treatment alone, is a feasible primary treatment for CC, however since it was unable to completely eradicate cells and a significant amount of the 20% of these cells remained in an active proliferative state, secondary spread could still occur. Even though research has reported that PDT has emerged as an effective and tolerable treatment strategy for the control of primary CC, it still requires improvement in relation to investigating enhanced PDT treatments capable of activating specific immune responses to fully eradicate metastasis [The findings substantiated previous discussions whereby it was noted that with irradiation alone, the CBD 99.8% , Ramer et al. (2010) and Contassot et al. (2004), whereby CBD was capable of significant forms of late apoptotic cell death in HeLa cells which are capable of anti-tumor proliferation responses, to prevent their metastasis in CC [Nevertheless, the experimental groups of the HeLa cells which received 0.125 µM ZnPcSis in CC ,40,47.4 PS plus 0.5 µM CBD with laser irradiation should not affect the biological responses of normal human tissues when applied in combination for the treatment of CC post-PDT. Furthermore, these results suggest that the minor early apoptotic forms of cell death, which were reported in the cell death assays in normal WS1 fibroblasts caused no unwanted cytotoxic effects and supported previous discussions, stating that these incremental amounts of cells were probably in an autophagic state and were able to recover post-PDT. Nevertheless, even these insignificant forms of cellular damage could be overcome in future studies, and the overall PDT treatment outcomes of this study could be improved upon if this ZnPcS4 PS was conjugated to an active targeting nanocarrier, to ensure that its cellular absorption is only taken up by cancer cells [The overall findings from this quantitative study noted that this combinative response, when activated with laser irradiation, was not capable of any significant forms of anti-proliferative effects in normal human WS1 fibroblast cells, when compared to the cells-only control (which received no forms of treatment) . These rer cells . Commercially purchased CC cell line HeLa (Cellonex CAT SS1411) was used in this study as the CC-positive cell line, whereas WS1 human skin fibroblasts procured from the American Type Culture Collection (ATTC CRL-1502) were used for result comparison outcomes to represent normal human tissues. Cells were cultivated in a T25 culture flask containing supplemented pre-warmed media. 2 and 85% humidity. Once 90% confluent monolayers of the cells were detached from the flasks using TrypLE Select™, consequent cellular pellets were re-suspended in fresh culture media.HeLa cells were cultivated in Dulbecco’s modified eagle medium (DMEM), enriched with 10% fetal bovine serum (FBS), 4 mM sodium pyruvate, 4 mM L-glutamine, 2.5 g/mL amphotericin β, and 100 U penicillin 100 g/mL streptomycin solution. WS1 cells were cultivated in minimum essential media (EMEM), enriched with 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 10% FBS, 2.5 g/mL amphotericin β and 100 U penicillin 100 g/mL streptomycin solution. These re-constituted cell cultures were then incubated at 37 °C in 5% CO5 cells/mL and WS1 cells at 3 × 105 cells/mL of supplemented media into sterile 3.4 cm diameter cell culture plates. The plates were incubated for 4 h to allow for cellular attachment, prior to conducting experiments in the sterile tissue-culture-treated plates. Thereafter, HeLa cells were seeded at a density of 5 × 104 PS PDT individual dose response assays, and combinative experiments. After 4 h, HeLa and WS1 culture plates were divided into the various control and experimental groups for CBD or ZnPcS4 PS or CBD alone, had these concentrations added to their fresh media. All culture plates were then re-incubated for an additional 20 h. Then, the PDT experimental and control groups were irradiated in the dark using a Roithner 1000 mA 673 nm high-power semiconductor diode laser, at a fluency of 10 J/cm2 for average time of 16 min and 8 sec [The cell- and irradiation-only control groups had their media simply replaced with fresh media. The control or experimental groups which needed varying or concentrations of ZnPcS4) PS used in this study was purchased from Santa Cruz® Biotechnology   . It exhibits three major Q-bands at emissions of 583, 634, and 674 nm, within the far-red spectral range, considered ideal for primary PDT cancer treatment [4 PS stock solution was diluted with 4 mL of 0.001 M PBS. This working stock concentration of ZnPcS4 PS was then further diluted in complete cell culture media, to acquire varying dose concentrations , which were utilized within the below discussed assays [The zinc (II) phthalocyanine tetrasulfonic acid (ZnPcS5 g/mol) a. ZnPcS4reatment . To makeCBD was administered to CC cells pre-PDT treatment. The 10 mg/mL CBD solution solubilized in 1 mL 99.8% ethanol, with a molecular mass of 314.46 g/mol, was commercially obtained from Sigma-Aldrich   b. The CB4 PS or CBD alone that could yield 50% cytotoxicity (ICD50), so that cellular viability could be ensured post-experimentation within combinative experimentation, to allow for biological result interpretations. Individual dose response studies were performed using the inhibitory concentration dose (ICD) method to determine the lowest concentration dose of ZnPcS4 PS in varying doses received it in its diluted form through their culture media. Four hours post incubation, control or experimental culture plate groups which required CBD varying doses or ICD50 concentrations received it in its diluted 99.8% ethanol form through their culture media. The culture plates were then re-incubated in the dark for an additional 20 h, after which the control and experimental groups which required irradiation received it. All culture plates had their media replaced and they were re-incubated again for 24 h before conducting LDH dose response assays. Following 4 h incubation, HeLa or WS1 cell control or experimental culture plate groups which required ZnPcS® non-radioactive cytotoxicity assay kit  was used to quantitatively measure lactate dehydrogenase (LDH), which is a cytosolic enzyme that is released upon cell lysis, that is directly proportional to cellular cytotoxicity. Briefly, 24 h post PDT, 50 μL of complete cell culture media supernatant from each experimental and control culture plate was removed and mixed with 50 μL of LDH reconstituted substrate mix in flat bottom 96-well microplates. To determine cytotoxicity from the LDH cellular lysis that was produced post treatment, LDH absorbance was measured at 490 nm, using a spectrophotometer .The CytoTox 9650 post PDT for ZnPcS4 PS and CBD was found to be 0.125 µM and 0.5 µM, respectively. Thus, 24 h post treatment, both of these concentrations were utilized in the below-combinative subcellular localization, flow cytometry cell death, and cellular proliferation assays, to determine the combined effects that ZnPcS4 PS and CBD had on CC and WS1 post treatment.The above dose response assays reported that the ICD50 0.125 µM ZnPcS4 PS and/or 0.5 µM CBD to accommodate for the required combinative PDT assays when both were applied in combination. The culture plates were then re-incubated in the dark for an additional 20 h. HeLa cells or WS1 cells were detached from cell culture flasks and seeded in culture plates, which had sterile cover slips inserted into their base at previously mentioned densities. After 4 h incubation to allow for cellular attachment of the coverslips, the complete growth medium from the culture plates was replaced. The culture plates were then divided into various experimental and control groups, and received varying volumes of complete cell culture media and pre-determined ICDv/v) formaldehyde solution was added for 10 min to allow for cellular fixation to the coverslips. Thereafter, 1 mL of rinsing solution consisting of ice-cold 0.01 M PBS in 1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) sodium azide buffer solution was briefly added and then discarded. The cells were then stained with 200 µL diluted primary antibody  for 30 min on ice and washed with rinsing solution. Then, the cells were stained with 200 µL diluted secondary antibody (5 µg/mL goat anti-mouse IgG-FITC: AB6785 AC Abcam) for 30 min on ice and washed with rinsing solution. Next, 50 µL of 1 µg/mL 40-6-Diamidino-2-phenylindole (DAPI) was added, and the culture plates were incubated for 5 min at room temperature, after which they were rinsed with 1 mL HBSS. The coverslips were then removed from the culture plates and attached to glass microscope slides with 50 µL of mounting medium. The slides were examined using the filter settings of a Carl Zeiss Axio Z1 Observer immunofluorescent microscope at 40× magnification. All experimentation was conducted in the dark, to ensure that the PS or fluorochromes used did not photo-bleach. Following incubation, the complete culture media in all culture plates were discarded, then placed on ice and rinsed with 1 mL ice-cold 0.01 M PBS. Then, 1 mL of 3.7%  was used for the detection and quantitation of cells undergoing early or late apoptosis, cells dying from necrosis cells, or cells remaining viable within combinative ZnPcSv/v) binding buffer to a 1 × 105 cells/mL. The samples were centrifuged, and 100 µL of each cellular suspension was stained with 5 µL of Annexin V-FITC solution and 5 µL of reconstituted propidium iodide staining solution. Before the acquisition, 400 μL of binding buffer 1× was added. For each sample, 20,000 events were acquired using a BD Accuri™ C6 flow cytometer, with a 488 nm solid-state laser (40 mW), and optimal photomultiplier (PMT) voltages were established for each channel. The matching BD Accuri C6 Plus Annexin V-FITC/PI software kit template was used for data acquisition. Debris and doublets were excluded from the analysis. Early and late apoptotic cells were identified for their positivity to Annexin V, and necrotic cells were identified for their positivity to PI, as well as live cells being identified as per non-stain controls, as shown in the FACS gating strategy represented in Briefly, with reference to ® luminescent cell viability assay (Promega™ PRG7571) was used to determine the number of metabolically active cells which can generate adenosine triphosphate (ATP) post irradiation. The assay utilizes the properties of a commercial thermostable luciferase, which generates a luminescent signal proportional to the amount of ATP released upon cell lysis, and so signifies their overall viability and abilities to recover/proliferate post treatment. The CellTiter-Glo4 PS and CBD PDT response assays was measured to determine the combinative anti-proliferative effects this treatment had on HeLa, as well as determine if there were any unwanted side effects on normal cells.The cellular proliferation of HeLa and WS1 cells post-irradiation for the various control and experimental groups of combinative ZnPcS3, multilabel high-performance multiplate counter (model 1420). The amount of ATP detected was quantified in direct proportion to the number of proliferating cells post treatment.Briefly, 100 μL of reconstituted reagent was added to an equivalent volume of cell culture plates cell suspension post treatment. The contents were then pipetted into a 96-well white-walled microplate, and stirred together on a shaker for 2 min to induce cell lysis. The microplate was then incubated at room temperature for 10 min to allow for the luminescent signal to stabilize. The luminescent signal was determined in relative light units (RLUs) using a Perkin Elmer, Victort-test and one-way analysis of variance (ANOVA) were used for comparing two or more means of normally distributed data, whereas the Mann–Whitney test was used for non-normally distributed data to determine the statistical difference between the various control and experimental groups. The error bars on graphs display the mean standard error (SEM), and if an experimental or control group reported a SD higher than 1 within a sample repeat, it was repeated to ensure data accuracy and precise statistical comparisons. Statistical differences between the various control and experimental groups were determined. Values within the 95% confidence interval  were accepted as statistically different and represented graphically.Experimental assays were all performed in triplicate and repeated over three different days, and an average of these results was reported. Raw data were captured using Microsoft excel sheets and statistical analysis was done using the GraphPad Prism version 9 software to determine the mean, standard deviation (SD), standard error, and significant changes for the data when comparing the various control and experimental groups. The Student 4 PS, as well as determine that ZnPcS4 PS and CBD in cultured HeLa CC cells were cable of inducing late apoptotic forms of cell death, proposing that this combinative treatment approach could possibly effectively allow PDT to annihilate primary CC tumors, with CBD secondary tumor inhibition, as well as beneficially induce nominal effects on healthy cells. The findings of this study presented sufficient evidence to quantitively observe the in vitro subcellular localization efficiency of ZnPcS4 PS PDT and CBD treatment would require in vitro research in enhancing the uptake of PS and CBD via active targeting nanosystems [In relation to trying to address some of the shortfalls noted in PDT cancer therapeutic treatments, future research into this combinative ZnPcSosystems . This maosystems . These iosystems . In vivoosystems .1O2 to effectively kill tumor cells [2 consumption and other angiogenic factors [2O2 by GOx, as well as produced intracellular ROS under laser irradiation by MnPc [In addition, numerous studies have noted that the failure of PDT within in vivo and clinical applications is due to the hypoxic environment that solid tumors possess. This limits the amount of molecular oxygen available in a tumor’s microenvironment, when a PS is activated to produce sufficient ROS and or cells . However factors ,57. Zhu  factors . The stu factors . After u by MnPc . The ant by MnPc .2 nanoparticles. Due to the enhanced oxygen delivery of MnO2 (manganese dioxide)-mediated oxygen generation and reduced oxygen consumption induced by Bu-mediated mitochondrial dysfunction, the hypoxia tumor microenvironment was reversed, leading to enhanced ROS generation [Clinically, most treatments used for cancers induce higher expressions of programmed cell death ligand 1 (PD-L1), which impairs the efficacy of anti-cancer drugs , includineration . The selneration .In another study, researchers investigated the cascade two-stage tumor re-oxygenation and immune re-sensitization mediated by self-assembled albumin-sorafenib NPs for enhanced photodynamic immunotherapy . They de4 PS PDT and CBD combinative treatment approach to possible in vivo and clinical translations. However, it does raise hope for CC treatment. When considering the fundamentals of this study, there is still far more in vitro research required to bring this ZnPcS


Morus alba have been used in traditional medicine to treat muscle-related symptoms such as aches, numbness, and stiffness. Despite its clinical use in traditional medicine, its active compounds and mode of action have not yet been investigated. Therefore, we aimed to isolate the compounds from the twigs of M. alba and deduce active compounds, key gene targets, and mechanism of action against sarcopenia using network pharmacology analysis. Using various isolation techniques and spectroscopic methods, 43 phytochemicals, including 3 new flavonoids, were isolated and performed network pharmacology analysis. According to the computational-assistant analysis, 28 compounds, 9 genes, and the PI3K-Akt-mTOR signaling pathway were deduced as expected active compounds (EAC), key targets, and the main signaling pathway. To verify the predicted results, the cell proliferation activities of the EAC were evaluated. Especially, moracin E and M significantly increased by 130% (p < 0.001) and 57% (p < 0.05), respectively, which have more than 2- and 1.5-fold stronger effects compared to the control. Furthermore, both increased the expression level of proteins involved in the PI3K-Akt-mTOR signaling pathway and myogenic proteins, including myogenin and MyoD. This study demonstrated that moracin E and M exhibit cell proliferative effects on skeletal muscle cells through the PI3K-Akt-mTOR signaling pathway.Twigs of  It has been recognized as a disease by an International Classification of Diseases-10 code in 20162. Since then, the disease code has been assigned in many countries, including Germany and Australia4. Sarcopenia increases the risk of falls, and fractures, consequently, leading to functional dependence and disability6. All these changes reduce the quality of life of the elderly and increase the mortality rate. However, there is no treatment for sarcopenia officially approved by regulatory agencies such as the FDA and EMA. Therefore, research on the development of a treatment for sarcopenia is not only very important but also urgent.Sarcopenia is an age-related disorder characterized by progressive loss of muscle mass, strength, and functionMorus alba Linné is an important herbal resource, and its root barks, twigs, leaves, and fruits have been used as traditional medicines and dietary supplements to treat various diseases and symptoms8. Especially, twigs of M. alba have been used to treat aches, numbness in joints, and muscle stiffness in traditional Korean medicine. Moreover, its major components, prenylated flavonoids, coumarins, moracins, and stilbenes showed various pharmacological activities such as anti-inflammatory and anti-aging effects, suggesting the possibility that they may be active against sarcopenia10. Although natural products, such as M. alba, are an important source of therapeutic opportunities, their use may be limited due to technical barriers to getting detailed information for the use11. Identifying their active constituents is one of the most important steps in overcoming these limitations. However, the chemical complexity and diversity of natural products make investigating of active compounds very challenging. The recently emerged network pharmacology suggests an efficient approach to these difficulties13.Recently, herbal medicines and dietary supplements derived from natural products have been attracting attention as a way to prevent sarcopenia caused by various factors. M. alba. Network pharmacology has the advantages of overcoming the limitations of research on natural products by effectively deducing active compounds and their mode of action. Our previous studies have confirmed that this method is sufficiently effective and efficient for natural product research16. In this study, we isolated compounds from the twigs of M. alba and applied network pharmacology analysis to deduce the active compounds, key targets, and pharmacological mechanisms against sarcopenia. We further demonstrated in vitro effects of M. alba on cell proliferation of C2C12 skeletal muscle cells and validated its mechanism experimentally by western blot analysis. Our results suggest a novel therapeutic approach and perspective for treatment of sarcopenia using natural products.The present study applied network pharmacology to identify the muscle-related gene targets and signaling pathways involved in the action mechanism of 1, 5 and 10) along with forty known compounds were isolated −-[H2O] ion at m/z 447.1082 . The 1H NMR spectrum , 6.31  and 6.30  and a singlet signal at δ 6.10 . In addition, the spectrum displayed two singlets at δ 5.73  and 5.96 , a coupled doublets at δ 5.54  and 6.51 , and three methyl groups at δ 1.37  and δ 1.76 . These proton signals together structure with the 13C NMR ‐5‐hydroxy‐3‐methacryloyl‐8,8‐dimethyl‐4H,8H‐pyranochromen‐4‐one and named as morusalbalin A.Compound rum Fig.  showed tNMR Fig.  signals NMR Fig.  and 8 ofly Figs.  and 8. F5 was isolated as yellow amorphous powder, C24H22O7 as the molecular formula was determined by HR‐ESI‐MS spectrum for ([M+HCOO]−)-[H2O] ion at m/z 449.1214 . The 1H NMR , 6.29  and 6.31  and one singlet at δ 6.17 . The 13C NMR 18. The HMBC spectrum  substituent. Moreover, The 1H NMR spectrums of δ 3.28 , 5.10 , 1.53 , 1.50  assured that the presence of isopentene group. In the HMBC experiment, H‐1‴ showed correlations with C‐7/C‐8/C‐8a/C‐2‴; H‐2‴ with C‐1‴/C‐4‴/C‐5‴. These results suggested that the isopentene group located at C‐8 in ring A ‐5,7‐dihydroxy‐3‐methacryloyl‐8‐(3‐methylbut‐2‐en‐1‐yl)‐4H‐chromen‐4‐one and named as morusalbalin B.Compound NMR Fig.  showed tNMR Fig.  showed aum Figs.  and 8 sh A Figs.  and 8. T10 was a yellow amorphous powder. The molecular formula of 10 was determined to be C25H26O7 by HR‐ESI‐MS spectrum for [M−H]− ion at m/z 437.1606 . The 1H NMR , 6.36  and 6.34 ; two singlets at δ 6.50  and 6.37 ; and a 7″‐hydroxy‐3″,8″‐dimethylocta‐2″,8″‐dienyl group at δ 5.20 , 4.76 , 4.69 , 3.87 , 3.27 , 1.90 and 1.52 , 1.72 , and 1.60 . The 13C NMR spectrum showed 25 carbon signals, including two methyls, four methylenes, seven methines, and twelve quaternary carbons ‐(−)‐6‐apigenin (11)10. The HMBC spectrum , C-6 (δ 112.9) and C-7 (δ 163.6) that confirmed the 7″‐hydroxy‐3″,8″‐dimethylocta‐2″,8″‐dienyl group located at C-6 in ring A. Therefore, the structure of compound 10 was established as 2--5,7-dihydroxy-6--4H-chromen-4-one and named as morusalbalin C. Additionally, known compounds were identified by comparing with the spectral data of the previously reported studies (Result S1).Compound NMR Fig.  spectrumons Fig. . The aboum Figs.  and 8 sh16. The cut-off values of QED and OB for the selection of EAC were set to 0.3 or more and TRUE, respectively. Based on these cut-off values, 28 compounds were selected as EAC  network Fig. . By anal6, 9, 12‐15, 19‐23, 26, 30, 35 and 36) were significantly increased proliferation of myoblasts compared to non‐treated cells. However, the others did not affect cell proliferation, and several compounds  remarkably reduced cell proliferation. These results may be caused by cell toxicity due to three treatments of the compounds during the cell differentiation process. The cell proliferative effects of 14 compounds were evaluated using the 5‐bromo‐2′‐deoxyuridine (BrdU) assay to confirm their definitive skeletal muscle cell proliferation activity. All the tested compounds were evaluated at the final concentration of 25 μM. Among the tested compounds, compounds 12‐15, 21, 23 and 30 significantly increased proliferation of myoblasts  showed cell toxicity during the cell differentiation period. Among the proliferative compounds, moracin E (21) showed the highest cell proliferation activity and moracin M (23) which belongs to the same chemical classification as 21 also exhibited remarkable efficacy on cell proliferation. Therefore, we selected these moracin derivatives further evaluate their effects on skeletal muscle cell growth. Furthermore, we conducted an assessment of cell toxicity for Moracin E (21) and Moracin M (23) across a concentration range of 0, 0.3, 1, 3, 10, 30, and 50 μM, exposing cells for 4 h, 24 h, and 48 h  demonstrated negligible cell toxicity, with a maximum reduction in cell viability to 83%, surpassing the 75% threshold. In contrast, Moracin M (23) demonstrated slight cell toxicity at a concentration of 50 μM, reducing cell viability to 73%. However, concentrations below 30 μM showed no significant impact on cell viability. All compounds were dissolved in Dimethyl sulfoxide , and the control samples were treated solely with Dimethyl sulfoxide.Prior to evaluating the cell proliferation activities of EAC, their cytotoxicity was measured by the 3‐(4‐5‐dimethylthiazoly‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay. All the tested compounds did not exhibit a cytotoxic effects on C2C12 cells at the concentration of 25 μM Fig. . The EAC8 h Fig. . Moracin21) and moracin M (23). When C2C12 cells were cultured with moracin E (21) and moracin M (23) separately, there was an observed increase in the levels of p-PI3K (phosphorylated PI3K), p-AKT, and p-p70S6K proteins, as well as myogenic transcriptional factors MyoD and Myogenin, when compared to non-treated cells  were treated separately with concentrations of 5 μM and 25 μM for both moracin E  as EAC against sarcopenia. Most of them linked to the target genes such as AKT1, RPS6KB1, MTOR and IGF1R which showed correlation with PI3K-Akt-mTOR signaling pathway in the C-T-P network  showed muscle cell proliferative activities in both MTT and BrdU assays. Four are classified as flavonoids (12‐15), two as moracins  and one as a stilbene (30). Although previous research has demonstrated the molecular mechanisms of flavonoids and stilbenes on skeletal muscle health24, that of moracins has not been explored. Therefore, we focused on investigating the effects of moracins on skeletal muscle cell growth and the underlying mechanisms. To confirm whether our candidate compounds promote myogenic differentiation, we compared the expression level of each protein involved in the PI3K-Akt-mTOR signaling pathway and myogenic proteins using western blotting. Myogenin also known as MyoG is crucial in the early stages of myogenesis and myofiber development25 and MyoD is a myogenic regulator that occurs mainly during myocyte fusion26. These two important components of muscle health were both increased in cells treated with moracins  in July 2020. This sample was cultivated in Yeongcheon, Gyeongsangbuk-do, Korea. A voucher specimen (TMA-202007) was deposited at the Herbarium of College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Korea. The collection and purchasing of the plant material and related research complies with relevant institutional, national and international guidelines and legislation.The dried twigs of M. alba (18.0 kg) were extracted with 100% MeOH (5 L × 3 times) under sonication for 4 h at room temperature. After filtration and evaporation under reduced pressure, the MeOH extract (73.70 g) was suspended in water and successively partitioned with n‐hexane, chloroform (CHCl3) and ethyl acetate (EtOAc) to obtain CHCl3‐, EtOAc‐, and water‐soluble fractions, respectively. Compounds isolation was performed using diverse isolation techniques including, open column chromatography and prep-HPLC under various conditions .The dried twigs of 16. Briefly, SwissADME database27 was used to obtain physicochemical properties of the isolates. Then, drug-likeness and oral bioavailability were estimated by the Quantitative Estimate of Drug-likeness (QED)28 and Veber’s rule29 using obtained the physicochemical properties, and we selected expected active compounds (EAC). SwissTargetPrediction and GeneCards databases31 were applied to collect targets of expected active compounds and disease-related, and common targets were selected as potential targets. STRING database32 was used to get protein–protein interaction (PPI) of the potential targets. The potential targets PPI network was constructed and analyzed using Cytoscape (3.9.1) to choose key targets. Biological processes and signaling pathways involved in the key targets were analyzed and visualized using DAVID 2021 database33 and ImageGP34. Thereafter, an integrated network, expected active compounds-key targets-pathways network, was established and analyzed using Cytoscape.The network pharmacology was performed according to the methods described in our previously reported studiesMouse myoblast cell line C2C12 are  grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% penicillin‐streptomycin for 48 h until the confluency reached 70%. Culture media was changed to differentiation media containing DMEM with 2% Horse Serum (HS) and 1% antibiotics for 6 days with medium changes every other day.37. Throughout the differentiation period, once every two days, the medium was substituted with a differentiation medium  and the EAC were treated. Subsequently, cell proliferation was measured at the 540 nm absorbance; cell proliferation activity of the EAC‐treated cells was calculated and compared to 100% of non‐treated cells.Cell proliferation effect of the EAC on C2C12 skeletal muscle cells was measured by MTT assay according to the previous reported studiesA more reliable cell proliferation activity was evaluated by a 5‐bromo‐2′‐deoxyuridine (BrdU) assay for compounds exhibiting proliferative activity in the MTT assay. The BrdU cell proliferation assay is a non‐isotopic assay for the in‐vitro quantitative detection of newly synthesized DNA of actively proliferating cells. Briefly, after 48 h of the initial cell seeding with 5 × 104 cells per well (96 well plate), the growth medium (DMEM containing 10% FBS) was replaced with a differentiation medium (DMEM containing 2% HS) and treated compounds every other day during differentiation period. Finally, 6 days after the differentiation induction, cell proliferation activity was calculated by comparison with the absorbance at 450 nm of the standard solution of BrdU in the non‐treated cells.After differentiation, cells were washed with cold phosphate‐buffered saline buffer. Following, the cells are lysed with Radioimmunoprecipitation assay (RIPA) buffer  containing a duo cocktail  for protein isolation. Total proteins were separated by sodium dodecyl sulphated‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to nitrocellulose membrane. The membranes were obstructed using 5% bovine serum albumin in TBS‐T  and incubated overnight at 4℃ with individual primary antibody. Following wash procedure with TBS‐T, it was incubated in secondary antibodies which are conjugated with horse‐radish peroxidase for 1 h at room temperature. Protein bands were visualized using a chemiluminescent substrate  and captured using FusionCapt Advance software. Antibodies used for western blot were as follows: p-PI3K p85, p-AKT, p-mTOR, p-p70S6K, p-4E-BP1, MyoD, Myogenin, β-Actin, Goat anti-mouse IgG secondary antibody HRP conjugated, Goat anti-rabbit IgG secondary antibody HRP conjugated. For quantification, protein levels were normalized by β-Actin bands and analyzed using Image J software.Statistical significance was assessed through one‐way analysis of variance (ANOVA) and multiple comparisons with Bonferroni correction. Statistical significance was set at p < 0.05. All statistical analyses were carried out utilizing GraphPad Prism software (version 5).This research does not involve the ethics of human and animal experiments.The authors confirm that all methods were performed in accordance with the relevant guidelines in the “Supplementary Information.


O-Phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal step of selenocysteine (Sec) synthesis in archaea and eukaryotes. How the Sec synthetic machinery recognizes and discriminates tRNASec from the tRNA pool is essential to the integrity of the selenoproteome. Previously, we suggested that SepSecS adopts a competent conformation that is pre-ordered for catalysis. Herein, using high-resolution X-ray crystallography, we visualized tRNA-dependent conformational changes in human SepSecS that may be a prerequisite for achieving catalytic competency. We show that tRNASec binding organizes the active sites of the catalytic protomer, while stabilizing the N- and C-termini of the non-catalytic protomer. Binding of large anions to the catalytic groove may further optimize the catalytic site for substrate binding and catalysis. Our biochemical and mutational analyses demonstrate that productive SepSecS•tRNASec complex formation is enthalpically driven and primarily governed by electrostatic interactions between the acceptor-, TΨC-, and variable arms of tRNASec and helices α1 and α14 of SepSecS. The detailed visualization of the tRNA-dependent activation of SepSecS provides a structural basis for a revised model of the terminal reaction of Sec formation in archaea and eukaryotes. The comparatively lower pKa (5.2 versus 8.3) and redox potential (−488 mV versus −233 mV) of SeH render Sec fully ionized under physiological conditions (Sec) is embryonically lethal in mice (l-serine (Ser) or Cys compromises selenoenzyme activity and selenoprotein folding  is one of only two events in nature to expand the genetic code and incorporate a nonstandard amino acid into the proteome ,2. Thougnditions  and resinditions . In highnditions , iodothynditions , and Selnditions . The sys in mice , and rep folding . Moreove folding .Sec in all organisms. The cycle commences with a misacylation event during which a promiscuous seryl-tRNA synthetase (SerRS) attaches Ser to tRNASec (Sec (l-seryl-tRNASec kinase (PSTK) and O-phosphoseryl-tRNASec selenium transferase (SepSecS) to improve the efficiency of SeH substitution  acceptor-TΨC helix, resulting in an 8/5 fold in prokaryotes  and allowed to cool to room temperature and renature on the bench. For crystallization, the holoenzyme and complexes were assembled in the assembly buffer: 20 mM Tris, pH 8.0, 200 mM NaCl, 5% (v/v) glycerol, 10 μM PLP, and either 0.5 mM TCEP (native SepSecS) or 5 mM TCEP (SeMet-SepSecS). Crystals of the holoenzyme were grown with 4 mg/ml human SepSecS mixed with 1 mg/ml unacylated tRNASec in 0.36 M lithium citrate, 15% (w/v) PEG 3350, and 0.1 M sodium cacodylic acid titrated with 0.04 M HCl to pH 6.3. Crystals of the underivatized binary complex were grown using 2.5–7.5 mg/ml human SepSecS•tRNASec (with a 2-fold molar excess of SepSecS) in 0.24 M lithium citrate, 9–10% (w/v) PEG 3350, and 0.1 M sodium cacodylic acid titrated with HCl to a pH of 6.2–6.4. Crystals of the SeMet-derivatized binary complex were produced with 5.0 mg/ml of human SeMet-SepSecS•tRNASec (with a 2-fold molar excess of SepSecS) in 0.28–0.3 M ammonium acetate, 19.8% (v/v) MPD, and 0.1 M sodium citrate titrated with 0.057 M HCl to a pH of 5.5. For all setups, 1 μl of the protein or complex was mixed with 1 μl of reservoir buffer and crystals were grown at +12°C. After structure determination, we identified that the originally cloned SepSecS gene harbored a V491A mutation.Crystals were obtained by the vapor diffusion, sitting drop method in a 96-well plate format (Hampton Research). Prior to assembly, tRNASec crystals indicated that wavelength of 0.979439 Å was optimal for anomalous diffraction data collection. The X-ray diffraction data were processed in HKL-2000  ethylene glycol prior to X-ray exposure, and those obtained with MPD were cryoprotected using 30% (v/v) MPD. The diffraction data were collected at cryogenic temperatures at the Life Sciences Collaborative Access Team (LS-CAT) beamline at APS-ANL. For the SeMet complex, a Se fluorescence spectrum scan in SepSecS-tRNAHKL-2000 .Sec is bound to the tetramer in two alternative conformations  phasing based on SeMet. SHELX was used to determine the positions of 58  out of a possible 68 Se atoms  with continuum electrostatic calculations using the Adaptive Poisson-Boltzmann Solver (APBS) software package plugin  and solvent to 2.0 and 78.00, respectively.To improve visualization of α16, feature-enhanced maps (FEM) that minimize noise and model bias were calculated using Phenix . The elee plugin . BrieflyProtein stability and integrity of the WT and SepSecS mutants were evaluated by comparing thermal unfolding profiles generated by a Tycho instrument (NanoTemper Technologies). For sample preparation, all proteins were diluted to 1 mg/ml (17.3 μM) in 20 mM HEPES, pH 8.0, 150 mM NaCl, 5% (v/v) glycerol, and 0.05% (v/v) Tween-20. The diluted protein samples were spun for 5 minutes at 12000 rpm to pellet and remove any aggregated protein. Finally, samples were loaded into Tycho capillaries (NanoTemper Technologies) and analyzed in duplicate.To follow binding during MST, each SepSecS mutant was labeled using the Monolith Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper Technologies). The labeling reaction was performed according to the manufacturer's protocol. Briefly, 20 μM of protein was mixed with the dye (in the supplied buffer), keeping a dye-to-protein molar ratio of 3:1 and incubated in the dark for 30 min. Unreacted dye was removed with the supplied, dye-removal column equilibrated with 20 mM HEPES, pH 8.0, 150 mM NaCl, 5% (v/v) glycerol, and 0.05% (v/v) Tween-20. The protein concentration and degree of labeling were determined using UV/VIS spectrophotometry at 650 and 280 nm. A degree of labeling of ∼0.8–1 was typically achieved. Subsequently, bovine serum albumin (BSA) was added to the labeled protein to a final concentration of 0.4 mg/ml.Sec was heat denatured for 1 min at +90–95 °C  and allowed to cool to room temperature and renature on the bench. Dilution series were then prepared according to the MO.Control software-protocol (NanoTemper Technologies) generated from an estimated Kd. A series of 2-fold dilutions of tRNASec were prepared in 10 μl of MST assay buffer to yield a range of tRNASec concentrations. For each measurement, 10 μl of each ligand dilution was mixed with 10 μl of labeled SepSecS, which led to a working SepSecS concentration of 5 nM. After 10 min, the samples were loaded into Monolith NT.115 Premium Capillaries (NanoTemper Technologies). MST for WT SepSecS was measured using the Monolith NT.Automated (NanoTemper Technologies) using 15% LED power and medium MST power. All other measurements were performed on a Monolith NT.115Pico instrument (NanoTemper Technologies) at room temperature using 5% LED power and medium MST power.For the MST experiment, the labeled SepSecS was adjusted to 10 nM with MST assay buffer  glycerol, 0.05% (v/v) Tween-20, and 0.4 mg/ml BSA). Prior to complex assembly, tRNASec, the setup was adjusted to maximize the ligand concentration. A series of 2-fold dilutions of tRNASec were prepared in 20 μl, and then 18 μl of each ligand dilution was mixed with 2 μl of 50 nM labeled SepSecS. The tRNASec concentrations were input into the MO.Control software and run in Expert Mode, using 5% LED, medium MST power, and an MST on-time of 20 s. For all studied interactions, replicates (n = 3–6) from independently pipetted measurements were analyzed  using the signal from a 5 s MST on-time.For the R398A and R398E mutants of SepSecS and Mut5 of tRNASec, MST for a single set of capillaries was run at +24, +26, +28, +30, +32 and +34°C to determine the Kd for the same sample at each temperature. Replicates of five or six per species were run using 5% LED power and medium MST power and analyzed using an MST on-time of 5 s. From the temperature and the associated Kd value, we generated the corresponding van’t Hoff plot by plotting ln(Ka) versus 1/T  and entropy (ΔS°) of binding according to Equation  cells via the activity of the selenoenzyme, formate dehydrogenase (FDH) , chloramphenicol (34 μg/ml), 10 μM IPTG, 1 μM Na2MoO4, 1 μM Na2SeO3, 50 mM HCOONa and 0.5% (w/v) glucose. On a separate LB plate for downstream validation experiments, 250 μl of each undiluted culture was plated. Plates were incubated in an anaerobic chamber  with a gas mix of 90% N2, 5% H2, 5% CO2 for 24 h at +25°C. The next day, the LB top agar (0.75% (w/v) agar) was prepared and supplemented with 1 mg/ml benzyl viologen (BV), 250 mM HCOONa and 25 mM KH2PO4 (pH 7.0). For each assay plate, 10 ml of the supplemented top agar was poured on and gently distributed to cover the plate. To visualize the BV reduction, plates were imaged 30 min after the overlay with the top agar.The activity of WT and the human SepSecS variants was assessed by evaluating their ability to rescue the loss of SelA in Δse (FDH) . The daySec, or SeMet-derivatized SepSecS•tRNASec. Crystals of holo SepSecS diffracted to 2.25 Å, whereas the native and SeMet binary complex crystals diffracted to 2.32 and 2.07 Å resolution, respectively  unwinds and the segment encompassing residues 11–20 assumes a coiled conformation  in the active sites of all previous structures ,41,43 imtructure . HoweverSec complex, obtained under high-citrate concentrations, revealed that citrate bound to a similar site near the P-loop in both the catalytic and non-catalytic protomers  clashes with the placement of tRNASec in the catalytic protomer. Thus, positioning of tRNASec into the active site requires organization of the P-loop.Our 2.25-Å resolution structure of holo SepSecS possessed a phosphate ion bound to the P-loop Figure  in a dis Figures . An intr Figures . By contr Figure , presumaSec binding is a pre-requisite for ordering the P-loop into a catalytically competent state that accommodates entry of the CCA-end into the active site, while small ligand binding may additionally stabilize the active sites in SepSecS. Together the tRNA and ligand binding pockets could help the enzyme distinguish different steps in the reaction cycle.Based on our structural data, we propose that tRNASec illustrated that positively charged catalytic pockets in SepSecS are complementary to the negative charges on the tRNASec backbone and phosphoserine and selenophosphate ligands  and thermodynamic parameters (i.e. ΔH° and ΔS°) of complex formation.Surprisingly, sequence conservation of polar residues of α1 is weak . This lat in α14 , especiat in α14 . To corrSec with Kd of 134 nM  yielded a ΔH° of −64.65 ± 11.30 kJ/mol and ΔS° of −0.0869 ±0.0374 kJ/(mol•K)  Figure . These dc Figure .Thus, the thermodynamic data support a model in which a favorable enthalpy, derived from electrostatic interactions between the enzyme and tRNA, drives complex formation to overcome the cost of conformational stabilization.Sec complex formation, we sought to investigate the contributions of individual residues in helices α1 and α14 in tRNA binding. Consequently, we engineered a series of enzyme mutants  and evaluated their binding to tRNASec using MST. Our results showed that primarily positive and solvent-exposed side chains in these helices are important for tRNASec binding.After establishing that polar interactions mediate SepSecS•tRNATi) suggest they follow the same unfolding trajectory and adopt the same structure as the WT enzyme  weakened affinity by more than an order of magnitude, while substitution with Glu (R398E) abolished binding -based Escherichia coli complementation assay , α1 mutants of SepSecS reduced BV equally well as the WT enzyme, arguing that SepSecS can form a productive complex with tRNASec over a wide range of binding affinities  , as, asSec (r (vSer) .Sec, the binding of SepSecS to the 8/5-fold Mut3 was expected. However, the binding affinity was significantly lower (∼2 μM versus 134 nM) compared to WT tRNASec. Conversely, the SepSecS interaction with Mut5 tRNASec exhibited a binding curve with the right part of the curve trailing up with no plateau, indicating that SepSecS cannot specifically engage with Mut5  have 10 or more nucleotides . By contrast, nearly all α14 mutants exhibited impaired catalysis. This impairment concurs with structural data showing that conserved α14 residues deliberately engage the 73GCCA76-3′ end of tRNASec  and 8G9Z (for SeMet-SepSecS•tRNASec).The coordinates and structure factors are deposited in PDB with the accession codes 7L1T (for holo SepSecS), 7MDL (for SepSecS•tRNAgkad182_Supplemental_FilesClick here for additional data file.


Epigenetic alteration is a key feature that contributes to the progression of bladder cancer (BC) and long non-coding RNAs serve crucial role in the epigenetic modulation. This study was designed to explore the epigenetic regulation of LINC00592 in BC. LINC00592 expression in BC was examined. Then, LINC00592 was silenced in BC cell followed by cell behavior analyses using CCK-8, transwell, western blot, or flow cytometry. Potential downstream target of LINC00592 was explored using RNA pull-down assay and methylation of WIF1 was determined using methylated-specific PCR. In addition, WIF1 or/and LINC00592 were silenced in BC cells followed by cell behavior analyses to explore the regulation between them. Upregulation of LINC00592 was significantly detected in BC tissues and cells. In BC cells silencing LINC00592 suppressed the proliferation, migration, and epithelial-mesenchymal transitions (EMT), but enhanced apoptosis. Moreover, LINC00592 recruited DNMT1, DNMT3A, and DNMT3B to enhance WIF1 promoter methylation. In addition, WIF1 overexpression suppressed the proliferation, migration, as well as EMT, but enhanced apoptosis. Silencing WIF1 significantly attenuated the role of silencing LINC00592 in suppressing the proliferative, migratory, and EMT ability of BC cells, and increasing the apoptosis. LINC00592 promoted the growth and metastasis of BC via enhancing the promoter methylation of WIF1 and decreasing WIF1 transcription. The GLOLong non-coding RNA (lncRNA) is a type of transcript more than 200 nt in length but without protein coding ability . AccumulDNA methylation is a common mammalian cell modification characterized by adding a methyl group at the CpG island using DNA methyltransferases (DNMTs) . Global Here, the function of LINC00592 in BC was determined in this study. Following this, WIF1 was predicted as a methylation target of LINC00592 and the underlying mechanism was also explored. Based on the investigation, we hope to offer some new evidence for the detection and therapy of BC.22.1A total of 25 pairs of BC and matched non-tumor samples were collected from the First Hospital of Changsha who had undergone operation from June 2021 to September 2022. Once collected, the samples were rapidly stored in liquid nitrogen. All the enrolled patients did not receive neoadjuvant therapy before operation. This investigation was approved by the Ethic Committee of the First Hospital of Changsha and patients have signed the informed consent.2.2Normal bladder epithelial cell and human BC cell lines: 5,637, T24, EJ, as well as SW780 were purchased from American Type Culture Collection  and maintained in RPMI-1640  containing 10% fetal bovine serum  and 100 U/mL streptomycin–penicillin at 37°C in a humidity cell incubator.Plasmids of short hairpin negative control (sh-NC), sh-LINC00529, sh-WIF1, overexpression (oe)-NC, and oe-WIF1 were purchased from the Guangzhou RIBOBIO . When cell reached 80% confluency, plasmids as candidate were transfected into target cells, respectively, using lipofectamine 2000 (Invitrogen) as suggested. After 48 h of transfection, cells were gathered for transfected efficiency confirmation or the following investigations.2.3Total RNA of tissue or cell samples was extracted using Trizol reagent  as suggested. cDNA was synthesized using the Takara reverse transcription kit as per manufacturer’s instruction. Following this, the qRT-PCR was conducted using SYBR Green  on an ABI 7500 instrument . The qRT-PCR utilized premiers are summarized in 2.43 per well were seeded in a 96-well plate and cultured for 0, 24, 48, and 72 h. Then, 10 μL of CCK-8 regent was hatched with the cells in each well at 37°C for 1 h. Finally, the optical density of the cells in each well was detected at 450 nm with a microplate reader (Thermo Scientific).CCK-8 assay  was carried out to assess the proliferation of cells. First, cells at a density of 5 × 102.55 cells/mL medium without serum. Then, the upper chamber was used to seed cells with 1% FBS and the lower chamber was padded with 500 μL of 10% FBS medium. After 24 h incubation at 37°C, cells in the upper chamber were swabbed using a swab. Then, 4% paraformaldehyde was used to fix the cells on the chamber at room temperature for 15 min followed by 0.5% crystal violet  staining for 20 min. Finally, the cells were pictured and analyzed under a phase-contrast microscope .The 24-well transwell assay  was used to detect the migration ability. Briefly, cells were collected, calculated, and adjusted in the density of 1 × 102.6Samples were lysed by the radio immunoprecipitation assay (RIPA) lysis buffer  containing protease inhibitors at 4°C for 15 min. After 10 min of centrifugation at 4°C, the supernatants were harvested for quantification using the bicinchoninic acid method (Boster). Following this, the protein solution was subjected to 10% SDS-PAGE followed by electro-transferring onto a PVDF membrane. The membrane was blocked with 5% skim milk at indoor temperature for 0.5 h and incubated with the primary anti-WIF1 , MMP-2 , MMP-9 , Vimentin , E-cadherin , DNMT1 , DNMT3A , DNMT3B , and GAPDH  at 4°C for 12 h. Subsequently, membrane was hatched with goat anti-rabbit  or rabbit anti-mouse  secondary antibody for 1 h, and proteins were developed using the enhanced chemiluminescence kit (Millipore) followed by quantification using the Image J Software .2.7Apoptosis of cells was determined using the Beyotime Annexin-V apoptosis kit  as suggested by the manufacturer. After staining, cell samples were subjected to a Attune NxT flow cytometry  followed by a FlowJo V10 CL software  analysis.2.8TM Magnetic RNA-Protein Pull-Down Kit (Thermo) was used for RNA pull-down. Briefly, cell samples were lysed and the magnetic beads were hatched with the 3′-biotin-labeled LINC00592 probes. Following this, the beads were hatched with lysates at 4°C for RNA–RNA binding protein interaction. Then the interacted complexes were eluted, collected, and subjected to western blot assay.Pierce2.9The binding of LINC00592 to DNMTs proteins was examined using a RIP kit  according to kit protocol. Briefly, cells were rinsed with phosphate buffer solution and a same volume of RIPA was added to lysate the cells at 4°C for 5 min and centrifuged for 10 min. During centrifugation, 100 μL RIP wash buffer was added to 50 μL of bead, and hatched with 5 μg corresponding antibody  for 30 min at indoor temperature. Then, the magnetic beads-antibody complex was washed, re-suspended in 900 μL RIP wash buffer and incubated with 100 μL cell lysate at 4°C overnight. Subsequently, the magnetic bead–protein complex was harvested and separated by protease K buffer followed by RNA extraction and qRT-PCR.2.10DNA sample was isolated using the DNeasy tissue kit . After quantification, 1 μg of DNA was added to 100 μL of water containing 7 μL 3 M NaOH for denaturation at 37°C for 10 min. Then, 550 μL of sodium bisulfite solution provided in the kit was hatched with DNA solution at 50°C water bath for 16 h. Then, DNA samples were purified and amplified with methylated/unmethylated specific primers.2.11t-test or the one-way analysis of variance followed by Tukey’s post hoc test was used for comparison as it was appropriate. Significant difference was considered as P < 0.05.Statistical analyses were performed using GraphPad Prism 6.0  and data were presented using mean ± standard deviation. Student’s 33.1First, the expression of LINC00592 was detected by qRT-PCR. It was presented that LINC00592 was significantly enhanced in BC tumor samples in comparison with the adjacent tissues . Also, L3.2To reveal the role of LINC00592, LINC00592 was significantly silenced in 5,637 cells  followed3.3The lncATLAS analysis revealed that LINC00592 was located in the nucleus . To furt3.4For further exploration, WIF1 was significantly overexpressed in 5,637 cells  followed3.5To further investigate whether LINC00592 promoted BC cells’ malignant behavior by inhibiting WIF1, rescue experiments were performed. First, qRT-PCR analysis showed that the expression of WIF1 decreased significantly after silencing WIF1 . Moreove4BC is a common neoplasm of the urinary system with its the rapid progression and high rate of recurrence . AccordiAccumulated evidence suggested that lncRNAs serve critical functions in the pathogenesis of BC . LncRNA WIF1 is a member of the Wnt pathway and has been identified downregulating in several cancers, including prostate, bladder, lung, and breast cancer . Previou5Taken together, this study has documented that LINC00592 is significantly upregulated in BC and WIF1 promoter was markedly hypermethylated in BC. Mechanically, aberrant expression of LINC00592 enhances the promoter methylation of WIF1, thereby downregulating WIF1 expression and decreasing its effect on mitigating the proliferation and metastasis of BC. These findings provided some new evidence on understanding the progression of BC, and targeting the abnormal methylation of WIF1 might be considered as potential biomarker and therapeutic targets of human BC.