A new phenothiazine derivative inhibits human cancer through targeting master gene PELP1

A new phenothiazine derivative inhibits human cancer through targeting master gene PELP1 | 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 Research Article A new phenothiazine derivative inhibits human cancer through targeting master gene PELP1 Dan Huang, Jiarong Yang, Aiqun Shen, Qian Wu, Wenjing Li, Zhe Wu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7521996/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 Background/Objectives : Master gene locates on the key nodes of many signaling pathways implicating in many kinds of physiology and pathology conditions. PELP1 has been demonstrated to be a master gene and oncogene. However, target therapy to PELP1 is lack. Our aim is to find a new compound that has the potential to bind and specifically target to PELP1. Methods : In this study, a new phenothiazine derivative was synthesized using a phenothiazine basic bone and a targeting mitochondria cationic structure. Its anti-cancer effect was assessed in many tumor cell lines in vitro and the action mechanism was explored. Results: Compound A, a novel phenothiazine derivative, selectively targets mitochondria in cancer cells, inhibiting survival, migration, invasion, and colony formation while inducing apoptosis (not autophagy). It downregulates PELP1, suppressing PI3K/AKT/mTOR signaling, as confirmed by molecular docking and siRNA. PELP1 activation via saRNA partially reverses Compound A's effects. This highlights its potential as a targeted anticancer agent. Conclusions: In short, compound A, a new phenothiazine derivative can inhibit malignant biological behaviour of human cancer through targeting master gene PELP1. Phenothiazine derivative Mitochondria Targeted therapy Master gene PELP1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Cancer is a public health problem that threatened millions of patients. In 2025, 2,041,910 new cancer cases and 618,120 cancer deaths are projected to occur in the United States[ 1 ]. Although the death rate continues to fall through 2022, the incidence of cancer still ranks second only to cardiovascular and cerebrovascular diseases[ 1 ]. Overall cancer incidence has declined in men but has been persistently rising in women (Corn and Feldman). Finding effective treating methods for cancer is still a common topic of concern to people. Chemotherapy drugs are the mainstay for cancer treatment, however, low selectivity and toxicity to normal tissues limit the effect. It was implied that the difference in mitochondrial membrane potential ( ΔΨm ) detectable between normal and malignant cells is at least 60 mV[ 2 ]. Delocalized lipophilic cations (DLCs) are a family of compounds that accumulate in mitochondria driven by the negative transmembrane potential. Some DLCs are sensitive to the higher ΔΨm in malignant cells, and selectively accumulate in cancer cells’ mitochondria[ 3 ]. These kinds of molecules share a hydrophobic structure and a delocalized positive charge, which together allow DLCs to cross lipid membranes and favor their accumulation in the mitochondria, because of the negative transmembrane potential. However, despite their effective in vitro prevention of mitochondrial damage, lipophilic cations suffer from a major drawback. Charge accumulation into the matrix results in mitochondrial membrane depolarization, which may account for the toxicity of these compounds at concentrations > 10µM[ 4 ]. Members of the DLC family are structurally diverse, and their mitochondrial targets are quite different. Tri-phenyl‐phosphonium (TPP) is a member of DLC family, belonging to non‐toxic chemical moiety that functionally behaves as a mitochondrial targeting signal (MTS) in living cells. So, in this study we selected TPP to serves as a chemical mitochondrial targeting signal. Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) - a potential protooncogene regulates estrogen receptor (ER). Its expression is upregulated in hormone-dependent tumor such as breast cancer[ 5 , 6 ] and ovary cancer[ 7 , 8 ]. Recently, it has been demonstrated that PELP1 could play an oncogenic role in several hormone-independent tumors[ 9 – 11 ] and was a prognostic marker of poor survival. PELP1 acts as a coactivator of multiple nuclear receptors and transcription factors and scaffold proteins coupled to various proteins and play a key role in the process of cell cycle, ribosome biosynthesis, chromatin remodeling, DNA damage response, histone reading, and RNA splicing [ 12 ]. PELP1 can interacts with tumor-associated genes p53 [ 13 , 14 ], mTOR [ 15 ] and c-Src [ 8 ], so it also can be seen as a new oncogene. Chlorpromazine belongs to classic phenothiazine antipsychotics. Due to their comprehensive bioactivity, phenothiazine and its derivatives have a significant role in tumor research. As early as the 1950s, people began to pay attention to the anti-cancer effects of chlorpromazine. They can exert antitumor activity through several mechanisms, the most important of which are the processes of inducing apoptosis, such as the inhibition of DNA-repair mechanisms and signal transduction pathways [ 16 ], but their direct DNA-damaging and membrane-destabilizing effects are also outstanding [ 17 ]. It is also important to emphasize the ability of phenothiazines in inhibiting MDR (multi-drug resistance) tumor resistance [ 18 ], and their anti-angiogenesis effect [ 19 ]. However, there is still a space for elevating the anticancer effect of chlorpromazine, decreasing the unnecessary antischizophrenia action and side effect such as drowsiness. In fact, for a long time, researchers have been looking to improve the anti-cancer effects of chlorpromazine. For example, some researchers utilized laser irradiation to elevate the anti-cancer effect of chlorpromazine [ 20 ]. New compounds have also been synthesized using phenothiazine rings [ 21 , 22 ]. The prospect of structural modification of phenothiazine is very necessary and objective. 2. Materials and methods 2.1 materials 2-chlorophenothiazine were purchased from Hubei HongXinRuiYu Fine Chemical Industry. Triphenylphosphine (TPP)(code number: T818895), 1,4-dibromobutane (code number: D806893) and Dichlorosulfoxide (code number: T819486) were purchased from Macklin. Mitochondrial Membrane Potential and Apoptosis Detection Kit (code number: C1071S), BCA Protein Concentration Kit (Enhanced) (code number: P0010S), and Mito-Tracker Red CMXRos (code number: C1049) was purchased from Beyotime. Autophagy kit (code number: MAK138) was purchased from Sigma-Aldrich. Fetal bovine serum (code number: 10099141) was purchased from Gibco. All antibodies were purchased from ABclonal including PELP1(code number: A9026 ), mTOR (code number: A2445 ), Phospho-mTOR-S2481 (code number: AP0978 ), PI3K (code number: A0265 ), Phospho-PI3K (code number: AP0854 ), AKT (code number: A18120 ), Phospho-AKT (code number: AP0005 ), p53 (code number: A11232 ), Cytochrome C (code number: A0225 ), PARP (code number: A19596 ), caspase-3 (code number: A0214 ), BAX (code number: A2211 ), Bcl-2 (code number: A0208 ), HRP-labeled secondary antibody goat anti-mouse (code number: AS003 ), and HRP-labeled secondary antibody goat anti-rabbit (code number: AS014 ). Dulbecco’s Modified Eagle’s Medium (DMEM) (code number: SH30022.LS) and RPMI media (code number: SH30027.02) were purchased from HyClone. The PELP1 small activating RNA and PELP1 small interfering RNA came from Jima pharmaceuticals in China. 2.2 Compound A synthesis and characterization Step 1: Take 10 mmol of 1,4-dibromobutane, 2 mmol 2-chlorophenothiazine and 4–10 mmol sodium hydride into 10 ml of DMF, the mixture protected from light and stirred at room temperature for 24 hours. after the reaction is terminated, the solvent was evaporated by a rotary evaporator, and purified by silica gel column to obtain the target compound, a white viscous liquid, which had a molecular weight of 369 and a yield of 50%. the reaction formula is as shown (Fig. 1 A above). Step 2: 1 mmol of the obtained product of the step 1 and 2 mmol of triphenylphosphine were dissolved in 5 ml of toluene, and the reaction was stirred in an oil bath at 100 ° C protected from light for 24 hours. Once the reaction finished, the solvent was evaporated with rotary evaporator, and purified by silica gel column. Then, we obtained the target compound, a white solid, that chemical name was [4-(2-Chloro-4a,10a-dihydro-phenothiazin-10-yl)-butyl]-triphenyl-phosp-honium (compound A), which had a molecular weight of 553 and a yield of 50%; the reaction formula is as shown (Fig. 1 A below). 2.3 Molecular docking Molecular docking refers to the process in which drug or small molecules and protein macromolecules interact by mutual matching and mutual recognition[ 23 ]. The theoretical basis of molecular docking is:1 drug molecule produces pharmacological effects, need to be close to the target molecules, and take appropriate orientation, matching each other in the necessary parts. Complementarity of drugs and receptors includes steric complementarity, electrical complementarity, and hydrophobic complementarity. 2 large molecules and small molecules are adjusted by appropriate conformation to obtain a stable complex conformation. 3 The process of molecular docking is to predict the correct relative position, orientation and specific conformation of two molecules in the complex to determine whether the small molecule can interact with the target protein. We used molecular docking binding with specific vitro experiments to determine the affinity of a drug molecule to a target protein. Molecular docking procedure was described before. In brief, the structure of the ligand, i.e. our new synthetic phenothiazine derivative, was built and energy optimized using the Modeller 9.10 software. We used free AutoDock Toolkit developed by the Scripps Research Institute and Olson lab for docking experiments. All of the water molecules were removed before the experiments so that our experiments were performed under non-aqueous conditions. The primary ligand bound to the binding pocket was the chosen conformation for the active site. The picture was prepared using Pymol 1.2R2 version. 2.4 Cell culture Huh-7 (Liver cancer), MRMT-1 (breast cancer), C6 (Glioma cells), Lovo (Colon cancer), NCl-N87 and AGS (Gastric cancer), Hela (Cervical cancer) and H520 (Lung squamous cell cancer), H1975 (Lung adenocarcinoma) cells were cultured at 37°C with 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) or RPMI-1640 medium, respectively, supplemented with 10% -15% fetal bovine serum (FBS). 2.5 Identifying whether compound A can target mitochondria in cancer cells Using the MRMT-1 and AGS cell lines and Mito-Tracker Red CMXRos kit detected whether compound A can aggregate in mitochondria. Specific operation method according to the instruction. 2.6 MTT assay Exponentially growing cells (4–10×10 3 cells/well) were respectively seeded in 96-well plates including Huh-7, MRMT-1, C6, Lovo, NCl-N87, Hela, H520, AGS and H1975. Twenty four hours later, cells were incubated with various concentrations of compound A (0, 0.5, 1, 2, 4, 6, 8 and 10 µM). 1) In treated concentration study, all cells were respectively treated with compound A for 24 h; 2) In treated time study, the cell lines were respectively treated with compound A for 12 and 36 h including MRMT-1, AGS and Lovo cells. At the end of MTT assay, the data was collected according to the MTT experiment operation method. 2.7 Plate clone formation assay Exponentially growing cells (1000 cells/well) of MRMT-1, AGS and Lovo were seeded in 6-well plates overnight. These cells were incubated with various concentrations of compound A (0, 2 and 4 µM) for 7 days. Then, cells in 6-well plates were fixed and stained by 0.1% crystal violet, and then the number of clones containing more than 50 cells was counted and photographed under a microscope. Calculate the clone formation rate according to the formula: Clonal formation ratio (compared with the control group %) = number of clones at control group / number of clones at administration group × 100%. 2.8 Autophagy assay and Mitochondrial Membrane Potential and Apoptosis Detection Autophagy kit and the mitochondrial & membrane potential & apoptosis detection kit were used to detect autophagy and apoptosis of tumor cells treated with compound A. Exponentially growing cells of MRMT-1, AGS and Lovo (1.5×10 5 cells/well) seeded in 24-well plate with round coverslip overnight. Then, cells were treated with compound A (0, 2, 4 µM) for 24 h. At the end of assays, cells were washed 1–3 times with PBS after 24 h of incubation, and then experiments went on as manufacturer's instructions and the results were observed by Fluorescence microscope. 2.9 Transwell migration and invasion assays Migration study: Exponentially growing cell’s density was adjusted to 2.5 × 10 6 /ml with RPMI1640 without serum. 200µl cells solution were added into upper chamber and lower chambers were added 500 µl RPMI with 20% serum and compound A (0, 2 or 4 µM). After cultured for 24 hours, the inner surface of upper chamber was scrubbed, external membrane surface was soaked into the pre-cooling methanol for 15 min and then soaked into the 0.1% crystal violet for 15 min. Cells that migrated to external membrane surface were counted. Invasion study: Matrigel was coated on the inner surface of basement member, other procedure was same to migration study. After cultured for 24 hours, cells that invaded to external membrane surface were counted. 2.10 RNA transfection PELP1 small activating RNA (saRNA), non-targeting saRNA (scrambled or control saRNA), PELP1 small interfering RNA (siRNA) and non-targeting siRNA (scrambled or control siRNA) were both designed according to the literature ( Supplementary Table 1–2). Exponentially growing cells were seeded into 60 mm dishes for protein extraction and into 24-well plates for proliferation assay. siRNAs and saRNAs were respectively transfected to a final concentration of 100 or 200 nM using the Dharma FECT transfection reagent, according to the manufacturer’s recommendations. In brief, after cells entered into the exponential growth phase, a mixture including siRNA and siRNA-control, or saRNA and saRNA-control, transfect reagent, were incubation with target cells. About several days later, the target cells were harvested and western bloting assay were employed to test the silence or activating effect. The most efficient siRNA or saRNA was selected to perform the later experiment. 2.11 Protein extraction and Western blotting Cells that were treated with compound A at 0, 2 and 4 µM for 24 h were lysed and adjusted to same concentration. Proteins (30 µg protein) separated by 8–15% SDS-PAGE and then shifted to a polyvinylidene difluoride membrane at 4˚C. The membrane was blocked in Trisbuffered containing 5% bovine serum albumin and 1% Tween-20 at room temperature for 1-1.5 h, and then incubated with prepared primary antibodies overnight at 4˚C. Primary antibodies were diluted as follow: PELP1, 1:1000;SRC, 1:1000; phospho-SRC, 1:1000; PI3K, 1:1000; phospho-PI3K, 1:1000; AKT, 1:1000; phospho-AKT, 1:1000; mTOR, 1:1000; phospho-mTOR, 1:1000; PARP, 1:1000; caspase-3, 1:1000; Bcl-2, 1:1000; Bax, 1:1000; Cyto-C, 1:1000; p53, 1:1000; and GAPDH, 1:5,000. The following day, all membranes were soaked into proper secondary antibody (dilution, 1: 5,000) for 1 h at room temperature. Finally, the proteins were detected using enhanced chemiluminescent kit. 2.12 Statistical analysis All experimental data was analyzed by one-way analysis of variance using SPSS 17.0 software. and figures were generated using GraphPad Prism 6.0 software. Statistically variation was indicated by P < 0.05, ImageJ 2.0 software was used to semi-quantify western blotting images. All tests were repeated 3 times and final results were showed in the way of means ± standard deviation. 3. Results 3.1 Compound A characterization The characterization of Compound A see Table 1 . Table 1 The characteristics of compound A Appearance White solid powder 1H NMR (400 MHz, CDCl3) δ 8.09–7.41 (m, 15H), 7.19 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 7.5 Hz, 1H), 6.96 (dt, J = 15.1, 7.6 Hz, 3H), 6.87 (dd, J = 11.9, 3.8 Hz, 2H), 4.03 (t, J = 5.7 Hz, 2H), 3.76 (td, J = 13.0, 8.4 Hz, 2H), 2.42–2.11 (m, 2H), 1.77 (dd, J = 15.4, 7.7 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 146.43, 144.38, 133.32, 133.24, 132.34, 131.74, 131.71, 130.79, 130.70, 128.73, 128.61, 127.99, 127.60, 127.49, 125.11, 123.88, 123.03, 122.38, 115.89, 115.86, 46.77, 29.63, 28.91, 27.79, 27.66, 19.24, 19.20 Molecular weight 552 Purity About 99% (method: HPLC; chromatographic column 6.4*250mm 5µm XDB-C18; mobile phase water: methyl alcohol = 20:80) Note: the detailed information was outlined at supplement information. 3.2 Compound A cant target “malignant” mitochondria in cancer cells Through mitochondrial membrane potential and apoptosis detection assay, it was watched that mitochondria of cells was dyed with red fluorescence by mito-tracker red CMXRos kit (Fig. 2 left picture). The cell was only treated with compound A was dyed with light yellow fluorescence (Fig. 2 middle picture), which indicated that compound A can enter the cell. Then through the merge of those two picture, we can see the red and light yellow fluorescence almost overlapped (Fig. 2 right picture), which suggested that compound A can target mitochondria in cancer cells. 3.3 Compound A suppressed viability and plate clone formation of human cancer cells 1) In treated concentration study (MTT), comparing with control and 2-chlorophenothiazine group, compound A could obviously suppress the cell viability in those nine kinds cell lines and its inhibition ability increase as its concentration mounting. The IC 50 was outlined in the Table 2 (calculated by Prism 6.0 software log(inhibitor) vs. normalized response—Variable slope). Refer to those result and the fact that there were no significant different of the inhibition of compound A on different cell lines, we select AGS, MTMR-1, Lovo cell lines to do other experiments by randomness and select treated concentration as 0, 2, 4µM in other experiment (excluding plate colon formation assay). 2) In treated time study (MTT), when the treated concentration of compound A was same, the inhibition ability of compound A increased over time in those three cell lines. We selected 24 h as compound A treated time for cost reason (Fig. 3 A-D). 3) In the plate clone formation assay, (in addition to the concentration of 0.5 µM) the cell colonies of AGS and MRMT-1 cell were significantly inhibited by compound A, and both concentration of compound A showed inhibition on lovo cells forming cell colonies (Fig. 4 ). Table 2 The IC50 values of the nine cell lines inhibited by compound A Cell line IC50 NCl-N87 6.139 ± 1.345 µM Huh-7 5.58 ± 1.2765 µM MRMT-1 5.898 ± 0.4035 µM C6 5.436 ± 0.343 µM Hela 4.4618 ± 0.78226 µM H1975 4.353 ± 1.701 µM H520 4.1 ± 2.397 µM Lovo 3.786 ± 1.018 µM AGS 2.56 ± 0.705 µM 3.4 Compound A suppressed migration and invasion of tumor cells In cell migration assay, the cells that migrated into the external membrane were decreased as the compound A concentration mounting at those three cell line (Lovo, MRMT-1 and AGS). In cell invasion assay, the results were similar to the migration assay in those three cell lines that the compound A could significantly suppress the invasion of cells from the upper chamber to the external of membrane (Fig. 5 ). 3.5 Compound A inducing tumor cells apoptosis, rather than autophagy With the increase of compound A concentration, there were no autophagy cells that would be dyed with blue fluorescence by autophagy kit (Fig. 6A). This suggested that compound A did not induce autophagy on cancer cells. However, the cell number of apoptosis increased as the increase of compound A concentration and the mitochondrial membrane potential decreased as the increase of compound A concentration (Fig. 6B-D). This suggested that compound A can induce apoptosis on cancer cells, rather than autophagy. 3.6 Compound A downregulated master gene PELP1 related pathway First, we used molecular docking software to confirm the affinity of compound A for the PELP1 target. Same as our prediction, molecular docking tool suggested that compound A could target PELP1 (Fig. 7 A-B and Table 3 ). Then, we used western blot to confirm the change in the expression of related proteins of possible “drug targets” after administration. the expression levels of oncogene PELP1 as well as its downstream targets were measured. The expression levels of Caspase-3 (activated), Bax, P53, Cyto-C and PARP increased significantly in compound A group in those three cell lines. However, it was identified that compound A significantly decreased the expression of PELP1, Phospho-SRC, mTor, phospho-mTor, phospho-AKT, phospho-PI3K, and Bcl-2 in those three cell lines, compared with levels in the control groups, at the same time there was no noteworthy difference in the expression of SRC, AKT and PI3K (Fig. 8 A). When oncogene PELP1 was silenced, the proteins expression levels of Phospho-SRC, mTOR, phospho-mTOR, phospho-AKT, phospho-PI3K decreased at AGS cells (Fig. 8 B). However, when PELP1 was activated by saRNA in AGS & Lovo cells, it can partly offset the inhibitory capacity of compound A (Fig. 8 C-D). From these results, we can conclude that this new phenothiazine derivative can inhibit most solid human cancers through targeting master gene PELP1. Table 3 Results of performing molecular docking 4. Discussion The history of phenothiazine compounds can go back to the second half of the 19th century, when a German chemist, Heinrich August Bernthsen began to study the structure of methylene blue, which was first synthesized in 1876 by Heinrich Caro. In 1885, two years after Bernthsen first managed to produce phenothiazine, he also succeeded in describing the structure of methylene blue. At the same time, Paul Ehrlich began to investigate the possible therapeutic use of methylene blue in malaria infections, which he first published in 1891. Due to this, methylene blue was often prescribed for patients with malaria in the subsequent period. Research proved that phenothiazines could be the widely used in the 1930s and 1940s. The insecticidal [ 24 ], anthelmintic [ 25 ], and antibacterial [ 26 ] effects of the compound were detected; however, it was not widely used for these purposes. In the 1940s, another novel phenothiazine derivative, namely promethazine was brought to the forefront of attention; it was investigated by Paul Charpentier at the Rhone-Poulenc laboratory in Paris. It was shown that promethazine had an antihistamine effect, which was later used in the therapy of allergic diseases and in anesthesia [ 27 ]. Since then, some kinds of antipsychotic phenothiazine ramifications have been put into clinical use. Then, research conducted in the last couple of decades indicated that phenothiazine compounds could have an important role in other fields of medicine as well, including in cure of tumorous, infectious, or neurodegenerative diseases [ 28 ]. However, the heat of research on phenothiazines has declined in recent years. Quantities of reports have manifested that chemotherapeutic drug exert anti-proliferative ability by inducing apoptosis. Apoptosis is a classic programed cell death mode and many deregulated genes have the potential to inducing or prohibiting apoptosis. Mitochondrial dysfunction may lead to apoptosis. Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) is an ER coregulator that functions in nuclear as well as in extranuclear actions [ 29 ]. Deregulation of PELP1 expression is also found in several cancers, including breast, brain, and ovarian. PELP1 high expression correlates with poor prognosis [ 30 ]. Moreover, PELP1 is closely linked to several important kinases such as PI3K and AKT [ 31 ]. Research has shown that PELP1 knockdown reduced the magnitude of mTOR signaling and PELP1 overexpression promoted activation of the mTOR signaling [ 32 ]. These emerging findings suggest that the proto-oncogene PELP1 functions as a scaffolding protein with unknown enzymatic activity to promote tumor progress, so alternative means of targeting PELP1 oncogenic function are urgently needed, and our laboratory found that one of the targets of chlorpromazine is PELP1 [ 11 ]. In fact, cytoplasmic PELP1 may influence mytochondrial respiration [ 33 ], implying PELP1 expression can be found in mitochondrial. However, due to the lack of selectivity of chlorpromazine for tumor treatment, we have modified the phenothiazine structure and get compound A ([4-(2-Chloro-4a,10a-dihydro-phenothiazin-10-yl)-butyl]-triphenyl-phosp). We found that compound A can suppress the most solid tumor by MTT assay. Although we found one of its action targets was PEPLP1, we didn’t confirm its function on animal models like subcutaneous tumor formation model in nude mice. And the cell lines were used to explore its mechanism was randomness because there were on significant difference on its function for those 9 kinds cell lines, so we supposed the compound A can exert inhibition ability on different cell lines by some targets that most tumor have. In this study we just found one mechanism of compound A, but we look forward people who interest in this kinds compound can do further study on its mechanism and structure modification. Declarations Ethics approval and consent to participate Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was funded by grants from Hubei Province Key R & D Project (2022BCE011) for ZFN and Hubei Province Education Department Scientific Program (Q20192802) for QW, Hubei University of Science and Technology Pharmacy College project (2018-19XZY03) for ZFN, Hubei University of Science and Technology clinical medicine research project (LCZX201503) for FXL and (LCZX201518) for ZFN, Hubei University of Science and Technology diabetes project (zx1315 and zx1004) and developing project (2019-21GP04) for ZFN and (2019-20X018) for ZW, Hubei Province Education Department of science and technology project (B2018178) and China National Natural Science Funding (81902937) for YLS, Xianning city high-level talents selected founding for ZFN. Author Contribution ZFN, WJL, GT and LFX designed and revised the manuscript. WJL and ZFN wrote the manuscript，drew figures and created the tables. WJL and other authors participate in the experimental process. 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Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation. Cell Death Dis. 2011;2(7):e181. 10.1038/cddis.2011.62 . GajdÁcs M, NovÉ M, Csonka Á, Varga B, SanmartÍn C, DomÍnguez-Álvarez E, Spengler G. Phenothiazines and selenocompounds: a potential novel combination therapy of multidrug resistant cancer. Anticancer Res. 2020;40(9):4921–8. 10.21873/anticanres.14495 . Varga B, Csonka Á, Csonka A, Molnár J, Amaral L, Spengler G. Possible biological and clinical applications of phenothiazines. Anticancer Res. 2017;37(11):5983–93. 10.21873/anticanres.12045 . Udrea AM, Staicu A, Smarandache A, Andrei IR, Badea MA, Avram S, Pascu ML, Pirvulescu RA, Balas M. Enhancement of chlorpromazine efficacy in breast cancer treatment by 266 nm laser irradiation. Sci Rep. 2024;14(1):30329. 10.1038/s41598-024-82088-1 . 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Ohlow MJ, Moosmann B. Phenothiazine: the seven lives of pharmacology's first lead structure. Drug Discov Today. 2011;16(3–4):119–31. 10.1016/j.drudis.2011.01.001 . Vadlamudi RK, Kumar R. Functional and biological properties of the nuclear receptor coregulator PELP1/MNAR. Nucl Recept Signal. 2007;5:e004. 10.1621/nrs.05004 . Kefalopoulou Z, Tzelepi V, Zolota V, Grivas PD, Christopoulos C, Kalofonos H, Maraziotis T, Sotiropoulou-Bonikou G. Prognostic value of novel biomarkers in astrocytic brain tumors: nuclear receptor co-regulators AIB1, TIF2, and PELP1 are associated with high tumor grade and worse patient prognosis. J Neurooncol. 2012;106(1):23–31. 10.1007/s11060-011-0637-y . Vadlamudi RK, Manavathi B, Balasenthil S, Nair SS, Yang Z, Sahin AA, Kumar R. Functional implications of altered subcellular localization of PELP1 in breast cancer cells. Cancer Res. 2005;65(17):7724–32. 10.1158/0008-5472.CAN-05-0614 . Gonugunta VK, Sareddy GR, Krishnan SR, Cortez V, Roy SS, Tekmal RR, Vadlamudi RK. Inhibition of mTOR signaling reduces PELP1-mediated tumor growth and therapy resistance. Mol Cancer Ther. 2014;13(6):1578–88. 10.1158/1535-7163.MCT-13-0877 . Truong TH, Benner EA, Hagen KM, Temiz NA, Kerkvliet CP, Wang Y, Cortes-Sanchez E, Yang C-H, Trousdell MC, Pengo T, et al. PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER(+) breast cancer. Oncogene. 2021;40(25):4384–97. 10.1038/s41388-021-01871-w . Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Supplementarytable.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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7521996","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509723420,"identity":"8ed71d0c-9f30-45f8-9b44-586ea0753b5b","order_by":0,"name":"Dan Huang","email":"","orcid":"","institution":"The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Huang","suffix":""},{"id":509723421,"identity":"ef0864be-52b1-4884-afbe-423106598b51","order_by":1,"name":"Jiarong Yang","email":"","orcid":"","institution":"Surgery， General Hospital of Central Theater Command","correspondingAuthor":false,"prefix":"","firstName":"Jiarong","middleName":"","lastName":"Yang","suffix":""},{"id":509723422,"identity":"da62b9ee-9910-4557-899b-00f57540127c","order_by":2,"name":"Aiqun Shen","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Aiqun","middleName":"","lastName":"Shen","suffix":""},{"id":509723423,"identity":"99e64e8a-f368-4574-874e-eb28981975b1","order_by":3,"name":"Qian Wu","email":"","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Wu","suffix":""},{"id":509723424,"identity":"a7ec0590-fd8b-4863-9ca0-2e910f86aa5b","order_by":4,"name":"Wenjing Li","email":"","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Li","suffix":""},{"id":509723425,"identity":"87eb3a0b-a135-4b17-899a-282d372e1842","order_by":5,"name":"Zhe Wu","email":"","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Wu","suffix":""},{"id":509723426,"identity":"d024b180-c4f2-4215-b2a7-6087b509c948","order_by":6,"name":"Tao Gao","email":"","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Gao","suffix":""},{"id":509723427,"identity":"d1c33c24-befe-4325-ade8-a73e368889b7","order_by":7,"name":"Yanling Sun","email":"","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yanling","middleName":"","lastName":"Sun","suffix":""},{"id":509723428,"identity":"59d58ade-e6c1-449f-a413-daa6110572ca","order_by":8,"name":"Fuxing Liu","email":"","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Fuxing","middleName":"","lastName":"Liu","suffix":""},{"id":509723429,"identity":"e107da83-9913-4da4-be87-4de23f7c9a89","order_by":9,"name":"Jialin Huang","email":"","orcid":"","institution":"The Central Hospital of Enshi Tujia and Miao Autonomous Prefecture","correspondingAuthor":false,"prefix":"","firstName":"Jialin","middleName":"","lastName":"Huang","suffix":""},{"id":509723430,"identity":"8996b6f2-2bab-48b7-a7a4-6c83614fb9f9","order_by":10,"name":"Jinyan Xia","email":"","orcid":"","institution":"Huanggang Polytechnic College","correspondingAuthor":false,"prefix":"","firstName":"Jinyan","middleName":"","lastName":"Xia","suffix":""},{"id":509723431,"identity":"67b57e31-4267-45ff-b059-39949e27e03d","order_by":11,"name":"Zhifeng Ning","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDACZiBmbJDgB7EPfPxnI8fG3n6AKC2SDUDq4Ay2NGM+njMJhG1ibGAAaWE+zMN2KHGehIMBXtXm7LyHX/7cYSHBL91+4TAPz4H0NgmGBIYfFdtwarFs5kuzkDwjISE550zBwTkSd3LbpBsPMPacuY1Ti8FhHjMDwzaJOoMbOQkH3hg8y22TOZDAzNhGQEtim4SEPUgLT8LhdDaJBANCWowfHARqMZBIP3CQ58DhBGK0mDE2ArVI3MhhODizIc2wDRjIB/H65fwZ448/2+ok+GekP/7wscFGXr69/eCDHxW4tQABmwSE5kFExwF86oGA+QOEZn9AQOEoGAWjYBSMVAAAum5dol0S0rYAAAAASUVORK5CYII=","orcid":"","institution":"Hubei University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Zhifeng","middleName":"","lastName":"Ning","suffix":""}],"badges":[],"createdAt":"2025-09-03 02:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7521996/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7521996/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90819970,"identity":"d45dd06c-44ef-48bd-9275-d48259e4542e","added_by":"auto","created_at":"2025-09-08 14:04:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25252,"visible":true,"origin":"","legend":"\u003cp\u003eCompound A synthesis\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/509d52e036ab46ebbb619ea9.png"},{"id":90819973,"identity":"66dc2152-1a0a-43b3-ae0b-31c66b691e0f","added_by":"auto","created_at":"2025-09-08 14:04:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37000,"visible":true,"origin":"","legend":"\u003cp\u003eResults of mitochondrial probe experiments. The results demonstrated that compound A can locate at the mitochondrial of cancer cells. The left was the fluorescence map of compound A (Blue or green fluorescence). The middle was the mitochondrial probe ( red fluorescence ).The right was merge.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/f2ea2c5d990bad7cdb9f0878.png"},{"id":90820824,"identity":"6d931e7a-45b3-4d51-9ab5-378f41f018fb","added_by":"auto","created_at":"2025-09-08 14:12:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":73696,"visible":true,"origin":"","legend":"\u003cp\u003eCompound A had the potential to inhibit several cancer cells’ proliferation. A.Compound’s antitumor activity tested by MTT assay in cells of AGS, C6, H520, H1975, Hela, HuH-7, NCl-N87, MRMT-1, and Lovo. B Compound administration time screening in cells of AGS, MRMT-1 and Lovo detected at 12, 24 and 36 h. C was negative control and D were positive control. Viability of control group (not administered group) was set at 100%. *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/b5c82b6e28b788efc5449193.png"},{"id":90819972,"identity":"1c58359b-9db6-4efe-9ffd-670b62cc30e6","added_by":"auto","created_at":"2025-09-08 14:04:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52636,"visible":true,"origin":"","legend":"\u003cp\u003eCompound A prohibited single cell colony forming ability of cancer cells. A The results of Plate clone formation experiment. B a statistical chart of left results. Result of control group (not administered group) was set at 100%. ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/67b45f54d45240891806f350.png"},{"id":90820826,"identity":"7f4dfcf3-28b2-4f6b-83e5-d199bd162037","added_by":"auto","created_at":"2025-09-08 14:12:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":149148,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of migration and invasion experiment from transwell small chamber proved compound A’s inhibitory effect on cancer cells. The right histograms were a statistical chart of left results. Result of control group (not administered group) was set at 100%. ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/fb617eb6be8982af134cc6b7.png"},{"id":90819977,"identity":"ff6ee209-6a7c-4d23-a597-1c80fd1f6846","added_by":"auto","created_at":"2025-09-08 14:04:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78553,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Compound on Tumor cell apoptosis and autophagy and Preliminary Exploration of Their Mechanism suggested that compound A may lead to apoptosis, not autophagy. A Autophagy result. B Apoptotic result. Apoptotic cells were stained green by Annexin V, Live cell mitochondria were stained red by Mito- Tracker Red CMXRos and the nucleus were stained blue by Hoechst 33342.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/ba7a72a237498e187681e433.png"},{"id":90820830,"identity":"e8fa6092-5335-43bd-aa89-1b7f7ffa8d5e","added_by":"auto","created_at":"2025-09-08 14:12:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":48311,"visible":true,"origin":"","legend":"\u003cp\u003eResults generated by homology modeling software-Modeller 9.18 suggested that compound A can specifically bind to PELP1 protein. A for the cartoon map. B as a ligand-protein interaction site.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/72953e3a0f9c7bec032bbe6c.png"},{"id":90821100,"identity":"ed6a7ad8-c550-4937-8611-9f8600b0f17d","added_by":"auto","created_at":"2025-09-08 14:20:33","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":117389,"visible":true,"origin":"","legend":"\u003cp\u003eExperiment of exploring the anti-tumor mechanism of compounds suggested that compound A had the potential to regulate PELP1 associated proteins. A The expression of PELP1-associated proteins and apoptosis-associated proteins detecting by Western blot and histograms were semi‑quantifcation of western blotting. B Expression degree of PELP1, P-PI3K, mTOR, P-mTOR, AKT and P-AKT in cells of AGS transfected with PELP1-siRNA and transfected vector detecting dy Western blot and histogram was semi‑quantifcation of western blotting. C Expression degree of PELP1 in cells transfected with PELP1-saRNA and transfected vector detecting by Western blot. D Effects of the compound on viability of cancer cells of Lovo and AGS that transfected with PELP1- saRNA and transfected vector respectively was detected by MTT assay at 24 h. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/652a93ca8f2bcba27e53c9c3.png"},{"id":91688313,"identity":"2abd925c-8841-4436-974c-bf25673e853c","added_by":"auto","created_at":"2025-09-19 08:09:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1691054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/6f926996-1b00-45d2-89d3-a31357ef66e1.pdf"},{"id":90819976,"identity":"b472cde5-85bc-4f70-9577-98bb390df2d8","added_by":"auto","created_at":"2025-09-08 14:04:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":248454,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/21aaccb93b61df9823e035df.docx"},{"id":90820823,"identity":"cd51c5e1-b6e9-4b4b-b3be-999d0a955bb2","added_by":"auto","created_at":"2025-09-08 14:12:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12501,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7521996/v1/94bffdea4a9ad3d2811488ad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A new phenothiazine derivative inhibits human cancer through targeting master gene PELP1","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer is a public health problem that threatened millions of patients. In 2025, 2,041,910 new cancer cases and 618,120 cancer deaths are projected to occur in the United States[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although the death rate continues to fall through 2022, the incidence of cancer still ranks second only to cardiovascular and cerebrovascular diseases[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Overall cancer incidence has declined in men but has been persistently rising in women (Corn and Feldman). Finding effective treating methods for cancer is still a common topic of concern to people.\u003c/p\u003e\u003cp\u003eChemotherapy drugs are the mainstay for cancer treatment, however, low selectivity and toxicity to normal tissues limit the effect. It was implied that the difference in mitochondrial membrane potential (\u003cem\u003eΔΨm\u003c/em\u003e) detectable between normal and malignant cells is at least 60 mV[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Delocalized lipophilic cations (DLCs) are a family of compounds that accumulate in mitochondria driven by the negative transmembrane potential. Some DLCs are sensitive to the higher \u003cem\u003eΔΨm\u003c/em\u003e in malignant cells, and selectively accumulate in cancer cells\u0026rsquo; mitochondria[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These kinds of molecules share a hydrophobic structure and a delocalized positive charge, which together allow DLCs to cross lipid membranes and favor their accumulation in the mitochondria, because of the negative transmembrane potential.\u003c/p\u003e\u003cp\u003eHowever, despite their effective in vitro prevention of mitochondrial damage, lipophilic cations suffer from a major drawback. Charge accumulation into the matrix results in mitochondrial membrane depolarization, which may account for the toxicity of these compounds at concentrations\u0026thinsp;\u0026gt;\u0026thinsp;10\u0026micro;M[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Members of the DLC family are structurally diverse, and their mitochondrial targets are quite different. Tri-phenyl‐phosphonium (TPP) is a member of DLC family, belonging to non‐toxic chemical moiety that functionally behaves as a mitochondrial targeting signal (MTS) in living cells. So, in this study we selected TPP to serves as a chemical mitochondrial targeting signal.\u003c/p\u003e\u003cp\u003eProline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) - a potential protooncogene regulates estrogen receptor (ER). Its expression is upregulated in hormone-dependent tumor such as breast cancer[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and ovary cancer[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recently, it has been demonstrated that PELP1 could play an oncogenic role in several hormone-independent tumors[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and was a prognostic marker of poor survival. PELP1 acts as a coactivator of multiple nuclear receptors and transcription factors and scaffold proteins coupled to various proteins and play a key role in the process of cell cycle, ribosome biosynthesis, chromatin remodeling, DNA damage response, histone reading, and RNA splicing [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. PELP1 can interacts with tumor-associated genes p53 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], mTOR [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and c-Src [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], so it also can be seen as a new oncogene.\u003c/p\u003e\u003cp\u003eChlorpromazine belongs to classic phenothiazine antipsychotics. Due to their comprehensive bioactivity, phenothiazine and its derivatives have a significant role in tumor research. As early as the 1950s, people began to pay attention to the anti-cancer effects of chlorpromazine. They can exert antitumor activity through several mechanisms, the most important of which are the processes of inducing apoptosis, such as the inhibition of DNA-repair mechanisms and signal transduction pathways [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], but their direct DNA-damaging and membrane-destabilizing effects are also outstanding [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is also important to emphasize the ability of phenothiazines in inhibiting MDR (multi-drug resistance) tumor resistance [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and their anti-angiogenesis effect [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, there is still a space for elevating the anticancer effect of chlorpromazine, decreasing the unnecessary antischizophrenia action and side effect such as drowsiness. In fact, for a long time, researchers have been looking to improve the anti-cancer effects of chlorpromazine. For example, some researchers utilized laser irradiation to elevate the anti-cancer effect of chlorpromazine [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. New compounds have also been synthesized using phenothiazine rings [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The prospect of structural modification of phenothiazine is very necessary and objective.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 materials\u003c/h2\u003e\u003cp\u003e2-chlorophenothiazine were purchased from Hubei HongXinRuiYu Fine Chemical Industry. Triphenylphosphine (TPP)(code number: T818895), 1,4-dibromobutane (code number: D806893) and Dichlorosulfoxide (code number: T819486) were purchased from Macklin. Mitochondrial Membrane Potential and Apoptosis Detection Kit (code number: C1071S), BCA Protein Concentration Kit (Enhanced) (code number: P0010S), and Mito-Tracker Red CMXRos (code number: C1049) was purchased from Beyotime. Autophagy kit (code number: MAK138) was purchased from Sigma-Aldrich. Fetal bovine serum (code number: 10099141) was purchased from Gibco. All antibodies were purchased from ABclonal including PELP1(code number: A9026 ), mTOR (code number: A2445 ), Phospho-mTOR-S2481 (code number: AP0978 ), PI3K (code number: A0265 ), Phospho-PI3K (code number: AP0854 ), AKT (code number: A18120 ), Phospho-AKT (code number: AP0005 ), p53 (code number: A11232 ), Cytochrome C (code number: A0225 ), PARP (code number: A19596 ), caspase-3 (code number: A0214 ), BAX (code number: A2211 ), Bcl-2 (code number: A0208 ), HRP-labeled secondary antibody goat anti-mouse (code number: AS003 ), and HRP-labeled secondary antibody goat anti-rabbit (code number: AS014 ). Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) (code number: SH30022.LS) and RPMI media (code number: SH30027.02) were purchased from HyClone. The PELP1 small activating RNA and PELP1 small interfering RNA came from Jima pharmaceuticals in China.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Compound A synthesis and characterization\u003c/h2\u003e\u003cp\u003eStep 1: Take 10 mmol of 1,4-dibromobutane, 2 mmol 2-chlorophenothiazine and 4\u0026ndash;10 mmol sodium hydride into 10 ml of DMF, the mixture protected from light and stirred at room temperature for 24 hours. after the reaction is terminated, the solvent was evaporated by a rotary evaporator, and purified by silica gel column to obtain the target compound, a white viscous liquid, which had a molecular weight of 369 and a yield of 50%. the reaction formula is as shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA above).\u003c/p\u003e\u003cp\u003eStep 2: 1 mmol of the obtained product of the step 1 and 2 mmol of triphenylphosphine were dissolved in 5 ml of toluene, and the reaction was stirred in an oil bath at 100 \u0026deg; C protected from light for 24 hours. Once the reaction finished, the solvent was evaporated with rotary evaporator, and purified by silica gel column. Then, we obtained the target compound, a white solid, that chemical name was [4-(2-Chloro-4a,10a-dihydro-phenothiazin-10-yl)-butyl]-triphenyl-phosp-honium (compound A), which had a molecular weight of 553 and a yield of 50%; the reaction formula is as shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA below).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Molecular docking\u003c/h2\u003e\u003cp\u003eMolecular docking refers to the process in which drug or small molecules and protein macromolecules interact by mutual matching and mutual recognition[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The theoretical basis of molecular docking is:1 drug molecule produces pharmacological effects, need to be close to the target molecules, and take appropriate orientation, matching each other in the necessary parts. Complementarity of drugs and receptors includes steric complementarity, electrical complementarity, and hydrophobic complementarity. 2 large molecules and small molecules are adjusted by appropriate conformation to obtain a stable complex conformation. 3 The process of molecular docking is to predict the correct relative position, orientation and specific conformation of two molecules in the complex to determine whether the small molecule can interact with the target protein. We used molecular docking binding with specific vitro experiments to determine the affinity of a drug molecule to a target protein.\u003c/p\u003e\u003cp\u003eMolecular docking procedure was described before. In brief, the structure of the ligand, i.e. our new synthetic phenothiazine derivative, was built and energy optimized using the Modeller 9.10 software. We used free AutoDock Toolkit developed by the Scripps Research Institute and Olson lab for docking experiments. All of the water molecules were removed before the experiments so that our experiments were performed under non-aqueous conditions. The primary ligand bound to the binding pocket was the chosen conformation for the active site. The picture was prepared using Pymol 1.2R2 version.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell culture\u003c/h2\u003e\u003cp\u003eHuh-7 (Liver cancer), MRMT-1 (breast cancer), C6 (Glioma cells), Lovo (Colon cancer), NCl-N87 and AGS (Gastric cancer), Hela (Cervical cancer) and H520 (Lung squamous cell cancer), H1975 (Lung adenocarcinoma) cells were cultured at 37\u0026deg;C with 5% CO2, in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) or RPMI-1640 medium, respectively, supplemented with 10% -15% fetal bovine serum (FBS).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Identifying whether compound A can target mitochondria in cancer cells\u003c/h2\u003e\u003cp\u003eUsing the MRMT-1 and AGS cell lines and Mito-Tracker Red CMXRos kit detected whether compound A can aggregate in mitochondria. Specific operation method according to the instruction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 MTT assay\u003c/h2\u003e\u003cp\u003eExponentially growing cells (4\u0026ndash;10\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) were respectively seeded in 96-well plates including Huh-7, MRMT-1, C6, Lovo, NCl-N87, Hela, H520, AGS and H1975. Twenty four hours later, cells were incubated with various concentrations of compound A (0, 0.5, 1, 2, 4, 6, 8 and 10 \u0026micro;M). 1) In treated concentration study, all cells were respectively treated with compound A for 24 h; 2) In treated time study, the cell lines were respectively treated with compound A for 12 and 36 h including MRMT-1, AGS and Lovo cells. At the end of MTT assay, the data was collected according to the MTT experiment operation method.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Plate clone formation assay\u003c/h2\u003e\u003cp\u003eExponentially growing cells (1000 cells/well) of MRMT-1, AGS and Lovo were seeded in 6-well plates overnight. These cells were incubated with various concentrations of compound A (0, 2 and 4 \u0026micro;M) for 7 days. Then, cells in 6-well plates were fixed and stained by 0.1% crystal violet, and then the number of clones containing more than 50 cells was counted and photographed under a microscope. Calculate the clone formation rate according to the formula: Clonal formation ratio (compared with the control group %)\u0026thinsp;=\u0026thinsp;number of clones at control group / number of clones at administration group \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Autophagy assay and Mitochondrial Membrane Potential and Apoptosis Detection\u003c/h2\u003e\u003cp\u003eAutophagy kit and the mitochondrial \u0026amp; membrane potential \u0026amp; apoptosis detection kit were used to detect autophagy and apoptosis of tumor cells treated with compound A. Exponentially growing cells of MRMT-1, AGS and Lovo (1.5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) seeded in 24-well plate with round coverslip overnight. Then, cells were treated with compound A (0, 2, 4 \u0026micro;M) for 24 h. At the end of assays, cells were washed 1\u0026ndash;3 times with PBS after 24 h of incubation, and then experiments went on as manufacturer's instructions and the results were observed by Fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Transwell migration and invasion assays\u003c/h2\u003e\u003cp\u003eMigration study: Exponentially growing cell\u0026rsquo;s density was adjusted to 2.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/ml with RPMI1640 without serum. 200\u0026micro;l cells solution were added into upper chamber and lower chambers were added 500 \u0026micro;l RPMI with 20% serum and compound A (0, 2 or 4 \u0026micro;M). After cultured for 24 hours, the inner surface of upper chamber was scrubbed, external membrane surface was soaked into the pre-cooling methanol for 15 min and then soaked into the 0.1% crystal violet for 15 min. Cells that migrated to external membrane surface were counted.\u003c/p\u003e\u003cp\u003eInvasion study: Matrigel was coated on the inner surface of basement member, other procedure was same to migration study. After cultured for 24 hours, cells that invaded to external membrane surface were counted.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 RNA transfection\u003c/h2\u003e\u003cp\u003ePELP1 small activating RNA (saRNA), non-targeting saRNA (scrambled or control saRNA), PELP1 small interfering RNA (siRNA) and non-targeting siRNA (scrambled or control siRNA) were both designed according to the literature ( Supplementary Table\u0026nbsp;1\u0026ndash;2). Exponentially growing cells were seeded into 60 mm dishes for protein extraction and into 24-well plates for proliferation assay. siRNAs and saRNAs were respectively transfected to a final concentration of 100 or 200 nM using the Dharma FECT transfection reagent, according to the manufacturer\u0026rsquo;s recommendations. In brief, after cells entered into the exponential growth phase, a mixture including siRNA and siRNA-control, or saRNA and saRNA-control, transfect reagent, were incubation with target cells. About several days later, the target cells were harvested and western bloting assay were employed to test the silence or activating effect. The most efficient siRNA or saRNA was selected to perform the later experiment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Protein extraction and Western blotting\u003c/h2\u003e\u003cp\u003eCells that were treated with compound A at 0, 2 and 4 \u0026micro;M for 24 h were lysed and adjusted to same concentration. Proteins (30 \u0026micro;g protein) separated by 8\u0026ndash;15% SDS-PAGE and then shifted to a polyvinylidene difluoride membrane at 4˚C. The membrane was blocked in Trisbuffered containing 5% bovine serum albumin and 1% Tween-20 at room temperature for 1-1.5 h, and then incubated with prepared primary antibodies overnight at 4˚C. Primary antibodies were diluted as follow: PELP1, 1:1000;SRC, 1:1000; phospho-SRC, 1:1000; PI3K, 1:1000; phospho-PI3K, 1:1000; AKT, 1:1000; phospho-AKT, 1:1000; mTOR, 1:1000; phospho-mTOR, 1:1000; PARP, 1:1000; caspase-3, 1:1000; Bcl-2, 1:1000; Bax, 1:1000; Cyto-C, 1:1000; p53, 1:1000; and GAPDH, 1:5,000. The following day, all membranes were soaked into proper secondary antibody (dilution, 1: 5,000) for 1 h at room temperature. Finally, the proteins were detected using enhanced chemiluminescent kit.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll experimental data was analyzed by one-way analysis of variance using SPSS 17.0 software. and figures were generated using GraphPad Prism 6.0 software. Statistically variation was indicated by P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ImageJ 2.0 software was used to semi-quantify western blotting images. All tests were repeated 3 times and final results were showed in the way of means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Compound A characterization\u003c/h2\u003e\u003cp\u003eThe characterization of Compound A see Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe characteristics of compound A\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAppearance\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite solid powder\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1H NMR (400 MHz, CDCl3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eδ 8.09\u0026ndash;7.41 (m, 15H), 7.19 (t, J\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 7.07 (d, J\u0026thinsp;=\u0026thinsp;7.5 Hz, 1H), 6.96 (dt, J\u0026thinsp;=\u0026thinsp;15.1, 7.6 Hz, 3H), 6.87 (dd, J\u0026thinsp;=\u0026thinsp;11.9, 3.8 Hz, 2H), 4.03 (t, J\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H), 3.76 (td, J\u0026thinsp;=\u0026thinsp;13.0, 8.4 Hz, 2H), 2.42\u0026ndash;2.11 (m, 2H), 1.77 (dd, J\u0026thinsp;=\u0026thinsp;15.4, 7.7 Hz, 2H).\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13C NMR (101 MHz, CDCl3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eδ 146.43, 144.38, 133.32, 133.24, 132.34, 131.74, 131.71, 130.79, 130.70, 128.73, 128.61, 127.99, 127.60, 127.49, 125.11, 123.88, 123.03, 122.38, 115.89, 115.86, 46.77, 29.63, 28.91, 27.79, 27.66, 19.24, 19.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMolecular weight\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e552\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePurity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbout 99% (method: HPLC; chromatographic column 6.4*250mm 5\u0026micro;m XDB-C18; mobile phase water: methyl alcohol\u0026thinsp;=\u0026thinsp;20:80)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote: the detailed information was outlined at supplement information.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Compound A cant target \u0026ldquo;malignant\u0026rdquo; mitochondria in cancer cells\u003c/h2\u003e\u003cp\u003eThrough mitochondrial membrane potential and apoptosis detection assay, it was watched that mitochondria of cells was dyed with red fluorescence by mito-tracker red CMXRos kit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e left picture). The cell was only treated with compound A was dyed with light yellow fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e middle picture), which indicated that compound A can enter the cell. Then through the merge of those two picture, we can see the red and light yellow fluorescence almost overlapped (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e right picture), which suggested that compound A can target mitochondria in cancer cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.3 Compound A suppressed viability and plate clone formation of human cancer cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003e1) In treated concentration study (MTT), comparing with control and 2-chlorophenothiazine group, compound A could obviously suppress the cell viability in those nine kinds cell lines and its inhibition ability increase as its concentration mounting. The IC 50 was outlined in the Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (calculated by Prism 6.0 software log(inhibitor) vs. normalized response\u0026mdash;Variable slope). Refer to those result and the fact that there were no significant different of the inhibition of compound A on different cell lines, we select AGS, MTMR-1, Lovo cell lines to do other experiments by randomness and select treated concentration as 0, 2, 4\u0026micro;M in other experiment (excluding plate colon formation assay). 2) In treated time study (MTT), when the treated concentration of compound A was same, the inhibition ability of compound A increased over time in those three cell lines. We selected 24 h as compound A treated time for cost reason (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). 3) In the plate clone formation assay, (in addition to the concentration of 0.5 \u0026micro;M) the cell colonies of AGS and MRMT-1 cell were significantly inhibited by compound A, and both concentration of compound A showed inhibition on lovo cells forming cell colonies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe IC50 values of the nine cell lines inhibited by compound A\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCell line\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIC50\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNCl-N87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.139\u0026thinsp;\u0026plusmn;\u0026thinsp;1.345 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHuh-7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2765 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMRMT-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.898\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4035 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5.436\u0026thinsp;\u0026plusmn;\u0026thinsp;0.343 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHela\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.4618\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78226 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH1975\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.353\u0026thinsp;\u0026plusmn;\u0026thinsp;1.701 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eH520\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.397 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLovo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.786\u0026thinsp;\u0026plusmn;\u0026thinsp;1.018 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAGS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.705 \u0026micro;M\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Compound A suppressed migration and invasion of tumor cells\u003c/h2\u003e\u003cp\u003eIn cell migration assay, the cells that migrated into the external membrane were decreased as the compound A concentration mounting at those three cell line (Lovo, MRMT-1 and AGS). In cell invasion assay, the results were similar to the migration assay in those three cell lines that the compound A could significantly suppress the invasion of cells from the upper chamber to the external of membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Compound A inducing tumor cells apoptosis, rather than autophagy\u003c/h2\u003e\u003cp\u003eWith the increase of compound A concentration, there were no autophagy cells that would be dyed with blue fluorescence by autophagy kit (Fig.\u0026nbsp;6A). This suggested that compound A did not induce autophagy on cancer cells. However, the cell number of apoptosis increased as the increase of compound A concentration and the mitochondrial membrane potential decreased as the increase of compound A concentration (Fig.\u0026nbsp;6B-D). This suggested that compound A can induce apoptosis on cancer cells, rather than autophagy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Compound A downregulated master gene PELP1 related pathway\u003c/h2\u003e\u003cp\u003eFirst, we used molecular docking software to confirm the affinity of compound A for the PELP1 target. Same as our prediction, molecular docking tool suggested that compound A could target PELP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B and Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Then, we used western blot to confirm the change in the expression of related proteins of possible \u0026ldquo;drug targets\u0026rdquo; after administration. the expression levels of oncogene PELP1 as well as its downstream targets were measured. The expression levels of Caspase-3 (activated), Bax, P53, Cyto-C and PARP increased significantly in compound A group in those three cell lines. However, it was identified that compound A significantly decreased the expression of PELP1, Phospho-SRC, mTor, phospho-mTor, phospho-AKT, phospho-PI3K, and Bcl-2 in those three cell lines, compared with levels in the control groups, at the same time there was no noteworthy difference in the expression of SRC, AKT and PI3K (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). When oncogene PELP1 was silenced, the proteins expression levels of Phospho-SRC, mTOR, phospho-mTOR, phospho-AKT, phospho-PI3K decreased at AGS cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). However, when PELP1 was activated by saRNA in AGS \u0026amp; Lovo cells, it can partly offset the inhibitory capacity of compound A (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). From these results, we can conclude that this new phenothiazine derivative can inhibit most solid human cancers through targeting master gene PELP1.\u003c/p\u003e\u003cp\u003eTable 3 Results of performing molecular docking\u003c/p\u003e\n\u003cp\u003e\u003cimg 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width=\"687\" height=\"209\"\u003e\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe history of phenothiazine compounds can go back to the second half of the 19th century, when a German chemist, Heinrich August Bernthsen began to study the structure of methylene blue, which was first synthesized in 1876 by Heinrich Caro. In 1885, two years after Bernthsen first managed to produce phenothiazine, he also succeeded in describing the structure of methylene blue. At the same time, Paul Ehrlich began to investigate the possible therapeutic use of methylene blue in malaria infections, which he first published in 1891. Due to this, methylene blue was often prescribed for patients with malaria in the subsequent period. Research proved that phenothiazines could be the widely used in the 1930s and 1940s. The insecticidal [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], anthelmintic [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and antibacterial [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] effects of the compound were detected; however, it was not widely used for these purposes. In the 1940s, another novel phenothiazine derivative, namely promethazine was brought to the forefront of attention; it was investigated by Paul Charpentier at the Rhone-Poulenc laboratory in Paris. It was shown that promethazine had an antihistamine effect, which was later used in the therapy of allergic diseases and in anesthesia [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Since then, some kinds of antipsychotic phenothiazine ramifications have been put into clinical use. Then, research conducted in the last couple of decades indicated that phenothiazine compounds could have an important role in other fields of medicine as well, including in cure of tumorous, infectious, or neurodegenerative diseases [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, the heat of research on phenothiazines has declined in recent years.\u003c/p\u003e\u003cp\u003eQuantities of reports have manifested that chemotherapeutic drug exert anti-proliferative ability by inducing apoptosis. Apoptosis is a classic programed cell death mode and many deregulated genes have the potential to inducing or prohibiting apoptosis. Mitochondrial dysfunction may lead to apoptosis. Proline, Glutamic acid-, and Leucine-rich Protein 1 (PELP1) is an ER coregulator that functions in nuclear as well as in extranuclear actions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Deregulation of PELP1 expression is also found in several cancers, including breast, brain, and ovarian. PELP1 high expression correlates with poor prognosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, PELP1 is closely linked to several important kinases such as PI3K and AKT [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Research has shown that PELP1 knockdown reduced the magnitude of mTOR signaling and PELP1 overexpression promoted activation of the mTOR signaling [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These emerging findings suggest that the proto-oncogene PELP1 functions as a scaffolding protein with unknown enzymatic activity to promote tumor progress, so alternative means of targeting PELP1 oncogenic function are urgently needed, and our laboratory found that one of the targets of chlorpromazine is PELP1 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In fact, cytoplasmic PELP1 may influence mytochondrial respiration [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], implying PELP1 expression can be found in mitochondrial.\u003c/p\u003e\u003cp\u003eHowever, due to the lack of selectivity of chlorpromazine for tumor treatment, we have modified the phenothiazine structure and get compound A ([4-(2-Chloro-4a,10a-dihydro-phenothiazin-10-yl)-butyl]-triphenyl-phosp). We found that compound A can suppress the most solid tumor by MTT assay. Although we found one of its action targets was PEPLP1, we didn\u0026rsquo;t confirm its function on animal models like subcutaneous tumor formation model in nude mice. And the cell lines were used to explore its mechanism was randomness because there were on significant difference on its function for those 9 kinds cell lines, so we supposed the compound A can exert inhibition ability on different cell lines by some targets that most tumor have. In this study we just found one mechanism of compound A, but we look forward people who interest in this kinds compound can do further study on its mechanism and structure modification.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was funded by grants from Hubei Province Key R \u0026amp; D Project (2022BCE011) for ZFN and Hubei Province Education Department Scientific Program (Q20192802) for QW, Hubei University of Science and Technology Pharmacy College project (2018-19XZY03) for ZFN, Hubei University of Science and Technology clinical medicine research project (LCZX201503) for FXL and (LCZX201518) for ZFN, Hubei University of Science and Technology diabetes project (zx1315 and zx1004) and developing project (2019-21GP04) for ZFN and (2019-20X018) for ZW, Hubei Province Education Department of science and technology project (B2018178) and China National Natural Science Funding (81902937) for YLS, Xianning city high-level talents selected founding for ZFN.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZFN, WJL, GT and LFX designed and revised the manuscript. WJL and ZFN wrote the manuscript，drew figures and created the tables. WJL and other authors participate in the experimental process. All the authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eAll data in this article can be got from correspondence under reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Kratzer TB, Giaquinto AN, Sung H, Jemal A. Cancer statistics, 2025. CA Cancer J Clin. 2025;75(1):10\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3322/caac.21871\u003c/span\u003e\u003cspan address=\"10.3322/caac.21871\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBegum HM, Shen K. 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Oncogene. 2021;40(25):4384\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41388-021-01871-w\u003c/span\u003e\u003cspan address=\"10.1038/s41388-021-01871-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Phenothiazine derivative, Mitochondria, Targeted therapy, Master gene, PELP1","lastPublishedDoi":"10.21203/rs.3.rs-7521996/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7521996/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground/Objectives\u003c/strong\u003e: Master gene locates on the key nodes of many signaling pathways implicating in many kinds of physiology and pathology conditions. PELP1 has been demonstrated to be a master gene and oncogene. However, target therapy to PELP1 is lack. Our aim is to find a new compound that has the potential to bind and specifically target to PELP1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: In this study, a new phenothiazine derivative was synthesized using a phenothiazine basic bone and a targeting mitochondria cationic structure. Its anti-cancer effect was assessed in many tumor cell lines in vitro and the action mechanism was explored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eCompound A, a novel phenothiazine derivative, selectively targets mitochondria in cancer cells, inhibiting survival, migration, invasion, and colony formation while inducing apoptosis (not autophagy). It downregulates PELP1, suppressing PI3K/AKT/mTOR signaling, as confirmed by molecular docking and siRNA. PELP1 activation via saRNA partially reverses Compound A's effects. This highlights its potential as a targeted anticancer agent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eIn short, compound A, a new phenothiazine derivative can inhibit malignant biological behaviour of human cancer through targeting master gene PELP1.\u003c/p\u003e","manuscriptTitle":"A new phenothiazine derivative inhibits human cancer through targeting master gene PELP1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-08 14:04:28","doi":"10.21203/rs.3.rs-7521996/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":"9fb41769-23b9-4f57-acd4-ccd292dd1183","owner":[],"postedDate":"September 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-19T08:08:38+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-08 14:04:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7521996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7521996","identity":"rs-7521996","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}