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Peiminine induces apoptosis in HepG2 cells. (A) HepG2 cells treated with peiminine were stained using DAPI solution and observed by fluorescence microscopy (×400). (B) Genomic DNA of HepG2 cells treated with peiminine for 24 h was assessed by agarose gel electrophoresis. (C) Annexin V-FITC/propidium iodide (PI) staining of HepG2 cells treated with peiminine for 24 h was determined by flow cytometry. (D) The apoptotic rate of HepG2 cells exposed to peiminine for 24 h was analyzed by flow cytometry. Data are presented as the mean±SD from three independent experiments. https://doi.org/10.1371/journal.pone.0201864.g002
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Peiminine is a compound isolated from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae family), which has demonstrated antitumor activities. But its precise molecular mechanism underlying antitumor activity remain elusive. In this study, peiminine-induced apoptosis towards human hepatocellular carcinoma and its molecular mechanism were inves...
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... Peiminine has been previously demonstrated to reduce the viability of HepG2 cells in a time-and dose-dependent manner and had an IC 50 of 4.58 lg/mL at 24 h. The result suggests that peiminine can induce apoptosis in human hepatocellular carcinoma HepG2 cells through extrinsic and intrinsic apoptotic pathways (Chao et al., 2019). Peiminine exhibits its anticancer activity by inhibiting glioblastoma in vitro and in vivo through cell cycle arrest and autophagic flux blocking (Zhao et al., 2018). ...
Fritillaria cirrhosa D. Don is a well-known medicinal plant of Kashmir Himalaya. Traditionally, it has been used to treat several diseases, including cancer. However, the molecular mechanism behind anticancer activity remains unclear. Therefore, in the present study, we have performed high performance- liquid chromatography-mass spectrometry (HR-LC/MS), network pharmacology, molecular docking and molecular dynamic (MD) simulation methods were used to explore the underlying molecular mechanism of F. cirrhosa for the treatment of breast cancer (BC). The targets of F. cirrhosa for treating BC were predicted using databases like SwissTargetPrediction, Gene Cards and OMIM. Protein–protein interaction analysis and network construction were performed using the Search Tool for the Retrieval of Interacting Genes/Proteins programme, and analysis of Gene Ontology term enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment was done using the Cytoscape programme. In addition, molecular docking was used to investigate intermolecular interactions between the compounds and the proteins using the Autodock tool. MD simulations studies were also used to explore the stability of the representative AKT1 gene peiminine and Imperialine-3-b-glucoside. In addition, experimental treatment of F. cirrhosa was also verified. HR-LC/MS detected the presence of several secondary metabolites. Afterward, molecular docking was used to verify the effective activity of the active ingredients against the prospective targets. Additionally, Peiminine and Imperialine-3-b-glucoside showed the highest binding energy score against AKT-1 (–12.99kcal/mol and −12.08kcal/mol). AKT1 with Peiminine and Imperialine-3-b-glucoside was further explored for MD simulations. During the MD simulation study at 100 nanoseconds, a stable complex formation of AKT1 þ Peiminine and Imperialine-3-b-glucoside was observed. The binding free energy calculations using MM/GBSA showed significant binding of the ligand with protein (DG: −79.83±3.0kcal/mol) between AKT1 þ Peiminine was observed. The principal component analysis exhibited a stable converged structure by achieving global motion. Lastly, F. cirrhosa extracts also exhibited momentous anticancer activity through in vitro studies. Therefore, present study revealed the molecular mechanism of F. cirrhosa constituents for the effective treatment of BC by deactivating various multiple gene targets, multiple pathways particularly the PI3K-Akt signaling pathway. These findings emphasized the momentous anti-BC activity of F. cirrhosa constituents.
... In recent years, phlegm-resolving CHMs have gradually received attention and been applied in the clinical treatment of PLC. Some of the above six phlegm-resolving herbs and their active ingredients or extracts have now been shown to have inhibitory effects on LC cells (Darvesh et al., 2012;XU et al., 2015;Chao et al., 2019). However, TCM treatment is a systemic process, and there are synergistic or antagonistic effects between various ingredients or their targets. ...
Objective: Nowadays, primary liver carcinoma (PLC) is one of the major contributors to the global cancer burden, and China has the highest morbidity and mortality rates in the world. As a well-known Chinese herbal medicine (CHM) prescription, Huatan Sanjie Granules (HSG) has been used clinically for many years to treat PLC with remarkable efficacy, but the underlying mechanism of action remains unclear.
Methods: A clinical cohort study was conducted to observe the overall survival of PLC patients with vs. without oral administration of HSG. Meanwhile, the BATMAN-TCM database was used to retrieve the potential active ingredients in the six herbs of HSG and their corresponding drug targets. PLC–related targets were then screened through the Gene Expression Omnibus (GEO) database. The protein–protein interaction (PPI) network of targets of HSG against PLC was constructed using Cytoscape software. The cell function assays were further carried out for verification.
Results: The results of the cohort study showed that the median survival time of PLC patients exposed to HSG was 269 days, which was 23 days longer than that of the control group (HR, 0.62; 95% CI, 0.38–0.99; p = 0.047). In particular, the median survival time of Barcelona Clinic Liver Cancer stage C patients was 411 days in the exposure group, which was 137 days longer than that in the control group (HR, 0.59; 95% CI, 0.35–0.96; p = 0.036). Meanwhile, the enrichment analysis result for the obtained PPI network consisting of 362 potential core therapeutic targets suggest that HSG may inhibit the growth of liver cancer (LC) cells by blocking the PI3K-Akt/MAPK signaling pathways. Furthermore, the above prediction results were verified by a series of in vitro assays. Specifically, we found that the expressions TP53 and YWHA2, the targets of the hepatitis B virus signaling pathway, were significantly affected by HSG.
Conclusion: HSG shows promising therapeutic efficacy in the adjuvant treatment of PLC.
... The anticancer effects of peiminine may also be related to its anti-inflammatory activity. Peiminine induces apoptosis in HepG2 cells to evade local inflammatory reactions and tissue damage (Chao et al., 2019). Peiminine downregulates the expression of inflammatory cytokines in cancer cells, decreasing the levels of COX-2, TNF-α, IL-1β, as well as suppressing NF-κB signaling (Lim et al., 2018). ...
Introduction: The clinical efficacy of Yiqi Sanjie (YQSJ) formula in the treatment of stage III colorectal cancer (CRC) has been demonstrated. However, the underlying antitumor mechanisms remain poorly understood.
Materials and methods: The aim of the present study was to comprehensively characterize the molecular and microbiota changes in colon tissues and fecal samples from CRC mice and in CRC cell lines treated with YQSJ or its main active component, peiminine. Integrative tandem mass tag-based proteomics and ultra-performance liquid chromatography coupled with time-of-flight tandem mass spectrometry metabolomics were used to analyze azoxymethane/dextran sulfate sodium-induced CRC mouse colon tissues.
Results: The results showed that 0.8% (57/7568) of all detected tissue proteins and 3.2% (37/1141) of all detected tissue metabolites were significantly changed by YQSJ treatment, with enrichment in ten and six pathways associated with colon proteins and metabolites, respectively. The enriched pathways were related to inflammation, sphingolipid metabolism, and cholesterol metabolism. Metabolomics analysis of fecal samples from YQSJ-treated mice identified 121 altered fecal metabolites and seven enriched pathways including protein digestion and absorption pathway. 16S rRNA sequencing analysis of fecal samples indicated that YQSJ restored the CRC mouse microbiota structure by increasing the levels of beneficial bacteria such as Ruminococcus_1 and Prevotellaceae_UCG_001. In HCT-116 cells treated with peiminine, data-independent acquisition-based proteomics analysis showed that 1073 of the 7152 identified proteins were significantly altered and involved in 33 pathways including DNA damage repair, ferroptosis, and TGF-β signaling.
Conclusion: The present study identified key regulatory elements (proteins/metabolites/bacteria) and pathways involved in the antitumor mechanisms of YQSJ, suggesting new potential therapeutic targets in CRC.
... Peiminine was further shown to reduce the expression of the long non-coding RNA LINC00659, which leads to upregulated miR-760 expression and ultimately inhibited colorectal cancer cell viability . Peiminine also decreases poly (ADP-ribose) polymerase (PARP) activity in hepatoma cells, and increases the expression of Bax, caspase-3, caspase-8, caspase-9, and cleaved PARP1, ultimately leading to apoptosis (Chao et al., 2019). It can also act as a chemosensitizer for doxorubicin in gastric cancer treatment by modulating the EGFR/FAK pathway (Tang et al., 2018). ...
Fritillaria cirrhosa D. Don and F. thunbergii Miq. belong to the genus Fritillaria within the Liliaceae family. They are used in traditional Chinese medicines that are often administered in clinical settings as they have notable effects on cough, bronchitis, pneumonia, lung injury, cancer, and other diseases. In this review, we focus on the history, origin, similarities, and differences in efficacy, chemical composition, and pharmacological outcomes of the drugs obtained from F. cirrhosa (FRC) and F. thunbergii (FRT). We list various valuable pharmacological effects of FRC and FRT, including antitussive, expectorant, anti-inflammatory, antioxidant, and anticancer effects. Thus, this review offers a basis for the medical application of and further research into the pharmacological impacts of these two drugs. We believe that new drugs derived from the phytoconstituents of F. cirrhosa and F. thunbergii that have specific therapeutic properties can be developed in the future.
... 09 microcapsules can cause ischemia and hypoxia in tumor tissue of liver cancer, and result in necrosis and apoptosis; medicated serum of B. paniculatum decoction can induce apoptosis of human lung cancer A549 and H1299 cells (Mi et al., 2021). Chao et al. (2019) discovered that peiminine isolated from B. paniculatum can induce apoptosis in human hepatocellular carcinoma HepG2 cells through both extrinsic and intrinsic apoptotic pathways. Modern pharmacological studies have demonstrated that TBMS I shown a powerful ability in inducing apoptosis; and it can induce apoptosis of A549, PC9, SKOV-3, CNE-2Z, BGC823, DU145, EC109, HL-60, SW480, U251, choriocarcinoma cells, etc. ...
Bolbostemma paniculatum (Maxim.) Franquet is a unique species in China with a long history of medicinal use, which has the effects of detoxifying, dissolving lumps and dispersing swellings. And it is commonly used to treat many diseases, such as carbuncle and sore, acute mastitis, mammary cancer, scrofula and subcutaneous nodule traditionally. Modern clinical studies have found that B. paniculatum and its compounds can be used for the treatment of a variety of cancers, mastitis, hyperplasia of mammary glands, chronic lymphadenitis, cervical lymph tuberculosis and surgical wart skin diseases, and the curative effect is positive. At present, a variety of Chinese patent medicines containing B. paniculatum have been exploited and marketed in China for the treatment of cancers, breast diseases and flat warts. This review article comprehensively discussed the traditional application, botany, chemical components, pharmacological activities, and quality control of B. paniculatum , put forward some noteworthy issues and suggestions in current studies, and briefly discussed the possible development potential of this plant as well as future research perspectives. 96 compounds have been isolated from B. paniculatum , including triterpenoids, sterols, alkaloids and other components, of which triterpenoid saponins are the main bioactive components. The crude extracts and monomer compounds of B. paniculatum have a wide range of pharmacological activities, such as anti-tumor, antiviral, anti-inflammatory, immunoregulatory, and so on. Moreover, its anti-tumor mechanism involves many aspects, including inhibiting cell proliferation, promoting cell apoptosis, blocking the cell cycle, interfering with cell invasion and metastasis, suppressing angiogenesis, and regulating autophagy. While there is a lack of systematic and in-depth research on its anti-tumor active components and mechanism of action at the moment; and a tight connection between the chemical composition and pharmacological activity of B. paniculatum has also not been established. Besides, a systematic quality determination standard for B. paniculatum should also be built, in order to carry out further research.
... Anti-cancer drugs often suppress cancer cells by apoptotic induction [25,26]. Apoptotic cells exposed several distinguishing characteristics such as cell shrinkage, the condensation of chromatin, nuclear DNA fragmentation, phosphatidylserine expression, and plasma membrane blebbing [27,28]. In this study, HepG2 cells maintained epithelial appearance in the low SFA-CHCl3 extract-treated groups. ...
... Cell cycle dysregulation is known to be characteristic of tumor cells. Anti-cancer drugs induce apoptosis by stopping the cell cycle progression in the G1, S, or G2/M stages [27,32,33]. In this study, flow cytometry demonstrated that the percentage of the G0/G1 phase of HepG2 cells under SFA-CHCl3 extract treatment decreased with the increase in time of the treatment, while the percentage of the G2/M phase increased. ...
The present study evaluated the effects of Sophora flavescens Ait. root extract on the proliferation of human hepatoma cell line HepG2. HPLC-UV analysis showed that the highest matrine and oxymatrine contents were obtained in the chloroform extract, compared to ethanol extract and ethyl acetate extract. The morphological analysis revealed that the chloroform extract of Sophora flavescens Ait. (SFA-CHCl3 extract) induced alterations of HepG2 cell morphology, resulting in the shrinkage of cells, the formation of debris, and cell detachment. The proliferation of HepG2 cells was inhibited by SFA-CHCl3 extract treatment. Cell cycle analysis exhibited that the cell proportion of the G0/G1 phase of HepG2 cells with SFA-CHCl3 extract treatment was decreased, while the cell proportion of the G2/M phase was increased. Flow cytometry analysis indicated a dramatic increase in the apoptotic percentage of HepG2 cells over the time of SFA-CHCl3 extract treatment. The SFA-CHCl3 extract also caused morphological changes in HepG2 nuclear, including chromatin condensation and DNA fragmentation. SFA-CHCl3 extract treatment induced the bax up-regulation and the bcl-2 down-regulation in HepG2 cells. These results revealed that SFA-CHCl3 extract could be a potential apoptosis inducer in HepG2 cells.
... Numerous drugs exert their antitumor functions by inducing cell apoptosis (Sun et al., 2004;Hu and Xiao, 2015). Peiminine induces apoptosis through both extrinsic and intrinsic apoptotic pathways in liver cancer, represses proliferation and growth of colorectal carcinoma by inducing autophagic cell death, and serves as an adriamycin chemosensitizer via the EGFR/FAK pathway in gastric cancer (Lyu et al., 2015;Tang et al., 2018;Chao et al., 2019). Similarly, in this study, the rate of OS cell apoptosis was significantly increased after peiminine treatment. ...
Aims: Peiminine has been reported to have various pharmacological properties, including anticancer activity. In this study, we investigated the effect of this alkaloid on osteosarcoma and explored the underlying mechanisms.
Methods: To evaluate the antiosteosarcoma effects of peiminine in vitro, cell viability was assessed by CCK-8 and live/dead assays; the effects of the drug on apoptosis and the cell cycle were examined by flow cytometry; the effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively, while its effects on autophagy were observed by transmission electron microscopy and an LC3 fluorescent puncta formation assay. The role of autophagy in the peiminine-mediated effects in osteosarcoma cells was evaluated by CCK-8 assay and western blotting after the application of the autophagy inhibitor chloroquine. The effect of peiminine on reactive oxygen species (ROS) production was analyzed using fluorescence confocal microscopy and spectrophotometry. Additionally, peiminine-treated osteosarcoma cells were exposed to SP600125, a JNK inhibitor, and N-acetylcysteine, a ROS scavenger, after which the contribution of the ROS/JNK signaling pathway to osteosarcoma was assessed using cell viability and LC3 fluorescent puncta formation assays, flow cytometry, and western blotting. A xenograft mouse model of osteosarcoma was generated to determine the antitumor effects of peiminine in vivo.
Results: Peiminine suppressed proliferation and metastasis and induced cell cycle arrest, apoptosis, and autophagy in osteosarcoma cells. These anticancer effects of peiminine were found to be dependent on intracellular ROS generation and activation of the JNK pathway. In line with these results, peiminine significantly inhibited xenograft tumor growth in vivo.
Conclusions: Peiminine induced G0/G1-phase arrest, apoptosis, and autophagy in human osteosarcoma cells via the ROS/JNK signaling pathway both in vitro and in vivo. Our study may provide an experimental basis for the evaluation of peiminine as an alternative drug for the treatment of osteosarcoma.
... We herein hypothesized that peiminine exerts similar inhibitory effects on osteoclastogenesis. Besides, many previous studies have reported that peiminine has certain anticancer effects (19,20) and is also effective against acute and chronic inflammatory injury (21)(22)(23)(24). In the context of orthopedic diseases, peiminine is known to protect articular cartilage from destructive cytokines (25), but its inhibitory effect on OCs and its effect on bone loss have not yet been reported. ...
Osteoclasts (OCs) play an important role in osteoporosis, a disease that is mainly characterized by bone loss. In our research, we aimed to identify novel approach for regulating osteoclastogenesis and thereby treating osteoporosis. Previous studies have set a precedent for screening traditional Chinese herbal extracts for effective inhibitors. Peiminine is an alkaloid extracted from the bulb of Fritillaria thunbergii Miq that reportedly has anticancer and anti-inflammatory effects. Thus, the potential inhibitory effect of peiminine on OC differentiation was investigated via a series of experiments. According to the results, peiminine downregulated the levels of specific genes and proteins in vitro and consequently suppressed OC differentiation and function. Based on these findings, we further investigated the underlying molecular mechanisms and identified the NF-κB and ERK1/2 signaling pathways as potential targets of peiminine. In vivo, peiminine alleviated bone loss in an ovariectomized mouse model.
... Despite the gradual progress of treatment, due to the late diagnosis of HCC, being prone to tumor recurrence and metastasis, and poor prognosis, the long-term overall survival time and outcome of HCC patients remain unchanged [15]. Apoptosis or programmed death plays a vital role in maintaining cell homeostasis and is considered to be the main mechanism of chemotherapeutic agents in eradicating cancer cells [16]. As we all know, HCC is a complex systemic disease that involves the interaction of multiple targets and signaling pathways; among them, the active antiapoptotic mechanism and metastasis in tumors may be the main reason for the failure of HCC treatment [17]. ...
Huxie Huaji (HXHJ) Ointment is a famous traditional Chinese medicinal prescription and is commonly used for the clinical treatment of hepatocellular carcinoma by boosting immunity and detoxification. However, the scientific evidence for the effect of HXHJ Ointment on hepatocellular carcinoma and the underlying molecular mechanism are lacking. The present study aimed to identify the effects of HXHJ Ointment on hepatocellular carcinoma in vitro and in vivo as well as investigating the mechanistic basis for the anticancer effect of HXHJ ointment. First, liquid chromatography-mass spectrometry was used to verify the composition of HXHJ Ointment and quality control. Second, in vitro, Cell Counting Kit (CCK8) cell viability assay and Hoechst 33342 staining assay were performed to explain the cell apoptosis. The protein levels of tumor suppressor protein (p53), B-cell lymphoma 2 gene (Bcl-2), cytochrome C (Cyt-C), and aspartate proteolytic enzyme-3 (caspase-3) were examined by immunofluorescence. Finally, in vivo, hematoxylin and eosin (H&E) staining was used to observe the pathological changes in hepatocellular carcinoma samples. Western blots and immunohistochemistry were used to detect the anticancer properties of HXHJ ointment. The results in vitro showed that 20% HXHJ Ointment serum could significantly inhibit HepG2 cell proliferation, increased tumor suppressor gene p53, downregulated antiapoptotic protein Bcl-2, promoted the release of mitochondrial Cyt-C, activated caspase-3, and induced HepG2 cell apoptosis. Furthermore, in vivo experiments showed that HXHJ Ointment could effectively inhibit tumor growth in nude mice xenotransplanted with HepG2 cells, changed the morphology of tumor cells, and regulated the expression of apoptosis-related protein pathway p53/Bcl-2/Cyt-C/caspase-3. HXHJ Ointment can significantly inhibit the development of hepatocellular carcinoma, and its mechanism may be related to the regulation of p53/Bcl-2/Cyt-C/caspase-3 signaling pathway to induce cell mitochondrial apoptosis.
1. Introduction
Hepatocellular carcinoma (HCC) is a more common malignancy than the majority of cancers and ranks second in the world’s top causes of cancer-related mortality [1]. Progress has been made in the diagnosis and therapy and we are encouraged by the recent success, while survival benefits remain modest due to the difficulty in being diagnosed early and the disease progressing quickly [2]. Currently, the major clinical treatment strategies for HCC include surgery, radiotherapy, and chemotherapy [3]. Unfortunately, radiotherapy and chemotherapy are not suitable for everyone and have the drawbacks of “kill a thousand enemies, hurt yourself eight hundred.” Moreover, those who have undergone hepatectomy or liver transplantation are frequently subject to survival declines due to recurrence and metastasis [4]. Thus, there is an urgent need for more effective treatment to reduce the rate of recurrence and metastasis, relieve clinical symptoms of HCC, and improve long-term patient survival and high quality of life.
Studies on looking for the new drugs that can kill tumor cells and enhance the body’s immunity may provide insight into novel therapeutic strategies. Traditional Chinese medicine (TCM), especially the formulas, has been used clinically for more than five thousand years in China and Asia. Various studies have shown that traditional Chinese medicine formulas (TCMF) have a marked effect on the treatment of liver cancer [5]. With an overall concept, it has a unique therapeutic effect and fewer side effects on HCC patients. It can not only inhibit tumor growth but also alleviate patients’ clinical symptoms and improve their quality of life [6].
Apoptosis is one such cell death pathway which allows a damaged or stressed cell to deconstruct in a regulated manner without causing inflammation and has garnered much attention in the cancer field, with hopes of eliminating malignant cells through its activation. Accumulating evidence has suggested that the mitochondrial pathway of apoptosis in a cell is one of the most important hallmarks of HCC [7, 8]. In mammals, activation of caspases is under the tight control of the Bcl-2 family proteins, named in reference to the first discovered mammalian cell death regulator [9, 10]. These proteins mainly act by regulating the release of caspases activators from mitochondria. Mitochondria appear today as the central executioner of apoptosis. Cyt-C is one of the apoptotic factors released by mitochondria into the cytoplasm and can bind to Apaf-1 (apoptosis protease activating factor) and induce cell apoptosis. The p53 tumor suppressor protein plays a central role in the regulation of apoptosis. Thus, documentation of changes to the expression of these markers would provide insights as to the mechanism whereby TCMF might be of value in the treatment of HCC.
Huxie Huaji (HXHJ) Ointment is a classic anticancer prescription and clinical studies have shown that HXHJ Ointment can improve patient quality of life and extend survival time when used to treat advanced primary liver cancer. It can significantly enhance the therapeutic effect and reduce tumor resistance in combination with Western Medicine chemotherapy. In our previous study, we found that HXHJ Ointment could significantly inhibit the proliferation of H22 cells by inducing the imbalance of the expression ratio of Bax and Bcl-2 proteins in vitro [11]. In the current study, our aim was to confirm the effects of HXHJ Ointment on HepG2 cells and tumor growth. Elucidation of the apoptosis mechanisms and molecular biology in HCC underlying HXHJ Ointment will provide more comprehensive and precise insight into clinical guidance.
2. Materials and Methods
2.1. Materials
Huxie Huaji (HXHJ) Ointment was made up of ten herbs, Codonopsis Radix, Astragali Radix, Herba Hedyotidis Diffusae, Agrimonia pilosa Ledeb., Scutellaria barbata D. Don., Reynoutria japonica Houtt., Smilax china L., Fructus Ligustri Lucidi, Buthus martensii Karsch, and Gekko swinhonis Guenther in the proportion 20 : 30 : 15 : 15 : 10 : 10 : 5 : 3 : 2 : 2. All the crude drugs were obtained from the Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine (Changsha, China). The voucher specimens were stored in the Herbal Medicine Room (Institute of Traditional Chinese Medicine, Hunan Academy of Traditional Chinese Medicine, Changsha). Cisplatin injection was purchased from Baxter International Co., Ltd. (Illinois, USA), with batch number 8H259A.
The CCK8 kit was purchased from Shanghai Qihai Biomedical Technology Co., Ltd. (Shanghai, China). DMEM/F12 medium and fetal bovine serum (FBS) were purchased from Gibco (New York, USA). Nuclear dye Hoechst 33258 and DAPI were purchased from Solarbio (Beijing, China). Bcl-2, caspase-3, Cyt-C, and p53 primary antibodies were purchased from Proteintech (Wuhan, China). GAPDH was purchased from ImmunoWay (New York, USA). Phosphodiesterase inhibitor, goat anti-mouse secondary antibody, and goat anti-rabbit secondary antibody were purchased from Auragene (Changsha, China). DAB color reagent kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China).
2.2. HXHJ Sample Preparation and Quality Control by Using HPLC and HPLC-Q-TOF
2.2.1. HXHJ Sample Preparation
According to the Chinese Pharmacopoeia (2015 edition), the HXHJ sample mixture was immersed in deionized water (6 times its total weight) overnight. Subsequently, they were decocted for 1 hour for the first time. Then, the extraction was repeated by adding deionized water (4 times its weight) and boiled for 1 hour. Finally, the two extracts were mixed. A part of the extract was concentrated and centrifuged to an appropriate concentration for HPLC-MS analysis, and the remaining part was freeze-dried for further use. The final amount of the extracted HXHJ powder was 230.34 g (yield, 14.28% (w/w)). The main pharmacologically active ingredients are known (Figure 1).
(a)
... Anticancer therapies that induce intracellular oxidative stress condition via the accelerated accumulation of ROS and / or interfere with the redox equilibrium through suppression of ROS elimination by inhibiting the antioxidant systems have been developed [74,75]. It is known that increased ROS levels cause damage to the cellular components (lipids, proteins, DNA), disturb the redox conditions and ultimately disrupt cell homeostasis. ...
... A 3 ml suspension of HepG-2 cells plated into 6-well plates (1 × 10 6 cells/well) was treated with compounds NFZ and 7 at concentrations of 0.278 and 0.08 μM, respectively, then incubated in humidified incubator at 37 • C with 5% CO 2 for 24 h. The manufacturer's instructions [74] were followed to determine caspase-3 activity. Cells were harvested and absorbance was measured by a spectrophotometer at λ 405 nm. ...
Drug repurposing has gained much attention as a cost-effective strategy that plays an exquisite role in identifying undescribed biological activities in clinical drugs. In the present work, we report the repurposing of the antibacterial drug nitrofurazone (NFZ) as a potential anticancer agent against CaCo-2, MDA-MB 231 and HepG-2 cancer cell lines. Novel series of nitrofurazone analogs was then designed considering the important pharmacologic features present in NFZ. Synthesis and biological evaluation of the target compounds revealed their promising anticancer activities endowed with antimicrobial potential and possessing better lipophilicity than NFZ. Compound 7, exclusively, inhibited the growth of all tested cancer cells more potently than NFZ with the least cytotoxicity against normal cells, displaying anti Gram-positive bacterial activities and antifungal potential. Analysis of the stereo-electronic properties of compound 7 via investigating the energies of HOMO, LUMO, HOMO-LUMO energy gap and MEP maps demonstrated its high reactivity and the expected molecular mechanism of action through reduction of the 5-nitrofuryl moiety. Data of the bioactivity studies indicated that the potent anticancer activity of 7 is mainly through increasing intracellular ROS levels and induction of apoptosis via significantly down-regulating the expression of Bcl-2 while up-regulating BAX, p53 and caspase 3 expression levels. Compound 7 potently inhibited the cellular expression levels of antioxidant enzymes GPx and GR compared to NFZ. Antioxidant enzymes kinetic studies and blind molecular docking simulations disclosed the mechanistic and structural aspects of the interaction between 7 and both GR and GPx. Thus, the successful discovery of 7 as a potential dual anticancer-antimicrobial nitrofurazone analog might validate the applicability of drug repurposing strategy in unravelling the unrecognized bioactivity of the present conventional drugs, besides furnishing the way towards more optimization and development studies.
