Nitidine Chloride Inhibits SIN1 Expression in Osteosarcoma Cells

Medicinal plant extracts have been widely used for cancer treatment. Nitidine chloride (NC) is a natural bioactive alkaloid that has recently been reported to have diverse anticancer properties. We aimed to investigate the cytotoxic effects of NC and the effectiveness of combinatorial treatment including NC and doxorubicin in breast cancer cells. Using MTT and flowcytometry assays, we found that NC induced cell growth inhibition and G2/M cell cycle arrest in a time- and dose-dependent manner both in MCF-7 and MDA-MB-231 breast cancer cell lines. Cancer cell growth inhibition was associated with increased levels of the p53 and p21 proteins. Apoptosis induction by NC treatment was confirmed by JC-1 mitochondrial membrane potential, annexin V-positive cell, and TUNEL staining. Using western blot analysis, we found that NC upregulated the pro-apoptotic proteins Bax, cleaved caspase-9 and -3 and cleaved PARP and that it downregulated the anti-apoptotic proteins Bcl-2 and PARP. By using the PI3K/Akt inhibitor LY294002, we further demonstrated that NC-induced apoptosis might be Akt-specific or dependent. In addition, NC exhibited a synergistic effect with doxorubicin on the growth inhibition of the human breast cancer cell lines MCF-7 and MDA-MB-231. Our study demonstrated the anticancer effect of NC on breast cancer and highlighted the potential clinical application of NC.

Metastasis is the main cause of death in osteosarcoma. Targeting the process of metastasis is a main strategy for osteosarcoma therapy. As a traditional Chinese medicine, Zanthoxylum nitidum (Roxb) has been applied to treat various diseases, including cancer. However, no evidence has been shown on the anti-metastasis effect of nitidine chloride (NC) that was extracted from Zanthoxylum nitidum (Roxb) on osteosarcoma cells, or its underling mechanisms. In the present study, we aimed to demonstrate the role of NC on the migration and invasion of osteosarcoma cells. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Then, by appling scratch wound healing assay and Transwell assays, we evaluated migratory and invasive ability of the cells, respectively. Moreover, the expression of epithelial-to-mesenchymal transition (EMT) markers were determined after treatment with NC. Furthermore, the expression of Akt, GSK-3β and Snail were detected by western blot analysis. In addition, the GSK-3β activity was examined by GSK-3β kinase assay. Finally, an inhibitor of GSK-3β, lithium chloride (LiCl) was applied to testify the effect of NC on the expression of EMT markers and Snail. We found that the proliferative, migratory and invasive ability of the U2OS osteosarcoma cells were all suppressed when treated with NC. NC increased the expression of E-cadherin and decreased the expression of N-cadherin, vimentin and fibronectin in a dose-dependent manner. NC also exerted its ability to suppress the phosphorylation of Akt and GSK-3β so as to activate GSK-3β. Then, by using an GSK-3β inhibitor, LiCl, we revealed the effect of GSK-3β in the expression of EMT markers. The expression of Snail was inhibited when treated with NC and LiCl also reversed the NC-inhibited Snail expression. Taken together, these results revealed that NC suppressed EMT and decreased the invasive ability of osteosarcoma cells via the Akt/GSK-3β/snail signaling pathway.

The anti-tumor action of engineered mesenchymal stem cells highlights the double-edged role of oncoproteins in osteosarcoma, and suggests the possibility of developing a novel strategy for protein-based cancer therapy.

The aim of the present study was to investigate the cytotoxic effects and underlying molecular mechanisms of nitidine chloride (NC) in hepatocellular carcinoma cells via quantitative proteomics. MTT assays were used to detect the inhibitory effects of NC in Bel-7402 liver cancer cells, and the number of apoptotic cells was measured by flow cytometry. Quantitative proteomics technology based on iTRAQ was used to discover differential expressed proteins after NC treatment, and bioinformatic techniques were further used to screen potential targets of NC. Molecular docking was applied to evaluate the docking activity of NC with possible upstream proteins, and their expression was detected at the mRNA and protein levels by quantitative reverse transcription PCR and western blotting. NC inhibited the proliferation of Bel-7402 cells after 24 h of treatment and stimulated apoptosis in vitro. The proteomics experiment showed that NC triggers mitochondrial damage in HCC cells and transcription factor AP-1 (c-Jun) may be a potential target of NC (fold change = 4.36 ± 0.23). Molecular docking results revealed the highest docking score of NC with c-Jun N-terminal kinase (JNK), one of the upstream proteins of c-Jun. Moreover, the mRNA and protein expression of c-Jun and JNK were significantly increased after NC treatment (p < 0.05). These findings indicate that NC significantly induced mitochondrial damage in HCC cells, and induced apoptosis by activating JNK/c-Jun signaling.

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

BackgroundNitidine chloride (NC) is a natural alkaloid that can inhibit tumor growth and induce apoptosis in varieties of cancers. However, the effec12/268t of NC on colon cancer (CC) cells has not been extensively studied.MethodsConlon cancer SW480 cells was treated with different concentrations of NC (0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 200 µM) in DMEM medium for 24 hours. Western blotting (WB) was used to detect the expression of related proteins, such as Ki67, PCNA, NANOG, SOX2, OCT4, Bcl-2, Bax, Caspase-3, Caspase-9, ERK1/2, p-ERK1/2, AKT, p-AKT, STAT3, p-STAT3, P65 and p-P65. The pellet formation experiment was used to detect the pellet formation of stem cells. The JC-1 experiment was used to detect the change of mitochondrial membrane potential. Kit was performed to detect the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA). In vivo experiments were used to verify the results of in vitro experiments. TUNEL assay was designed to detect the apoptosis in mice tissue. IHC was used to detect expression of Ki67 and OCT4 protein in tissue.ResultsNC significantly inhibited the expression levels of Ki-67 and a proliferating cell nuclear antigen (PCNA). NC can reduce the pellet colony and pellet size of tumor stem cells and block the stem cell characteristics of CC cells. The corresponding stem cell marker molecules NANOG, SOX2, and OCT4 were also downregulated. NC treatment induced the mitochondrial membrane potential depolarization of CC cells. The expression of pro-apoptotic proteins such as caspase-3, caspase-9, and Bax were upregulated, while the expression level of apoptotic Bcl-2 was significantly down-regulated. Moreover, NC reduced SOD activity and MDA content in CC cells. In addition, studies on pathway phosphorylation have shown that NC inhibits the expression of p-erk and p-akt proteins. Finally, the results were further confirmed by experiments in nude mice. NC inhibited tumor growth in mice. NC promoted apoptosis in tissues. NC inhibited the expression of Ki67 and OCT4 in tissues. NC inhibited the phosphorylation of pathway proteins ERK1/2 and AKT in tissues.ConclusionsNC treatment inhibited the proliferation and stemness of CC tissues, promoted the apoptosis of tumor tissues, downregulated the expression of p-ERK and p-AKT in tumor tissues, which suggests that NC may play an important role in regulating ERK and AKT pathways.

Nitidine chloride (NC) is a natural bioactive phytochemical alkaloid that has displayed anticancer activity in various types of cancer. However, no evidence has been reported for the direct effect of NC on CRC cell proliferation and apoptosis, and the underling mechanisms to be fully elucidated. The present study aimed to investigate the influence of NC on the apoptosis and proliferation of CRC cells. The viability and proliferation of CRC cells was measured by MTT assay and a [3H] thymidine uptake assay. Apoptosis was measured using a flow cytometric apoptosis assay and TUNEL staining. The expression levels of apoptotic-regulated proteins in addition to extracellular signal-regulated kinase (ERK) were measured by western blot analysis following stimulation with NC. The results indicated that NC inhibited the proliferation of HCT116 cells in a dose- and time-dependent manner. Additionally, apoptotic induction by NC treatment was confirmed. Furthermore, NC was demonstrated to significantly upregulate the expression of Bax, p53, cleaved caspase-3 and -9 and downregulate the expression of Bcl-2. Treatment with NC reduced the phosphorylation of ERK and by using an ERK inhibitor, U0126, the roles of NC in apoptotic induction and the inhibition of proliferation were further demonstrated. These results demonstrated that NC inhibited the proliferation and induced the apoptosis of CRC cells via the ERK signaling pathway.

Nitidine chloride (NC) exhibits anti-tumor properties in various types of tumor. However, to the best of our knowledge there is no previous evidence of NC involvement in the apoptosis or proliferation of ovarian cancer cells and the underlying molecular mechanisms. The present study aimed to investigate the influence of NC on the viability and apoptosis of ovarian cancer cells and the synergistic effect NC and doxorubicin (DOX) may have on ovarian cancer cells. The viability and proliferation of ovarian cancer cells were examined using a methyl thiazolyl tetrazolium assay and 3H-thymidine incorporation assay. The apoptotic rate of ovarian cancer cells was detected by flow cytometry. The expression of apoptosis‑associated proteins and Akt serine/threonine kinase1 (Akt) were determined by western blot analysis following NC treatment. The inhibitory effect of NC on the proliferation of ovarian cancer cells was demonstrated in a time and dose‑dependent manner. The pro-apoptotic effect of NC on ovarian cancer cells was also observed. It was determined that NC significantly downregulated the protein expression levels of B‑cell CLL/lymphoma 2 (Bcl-2) and upregulated the expression of Bcl‑2‑associated X protein, p53, caspase‑3 and ‑9. NC suppressed Akt phosphorylation. Additionally, the present study demonstrated that the effect of NC on the proliferation and apoptosis of ovarian cancer cells was Akt‑dependent by using the phosphatidylinositol-4,5-bisphosphate3-kinase/Akt signaling pathway inhibitor, LY294002. NC exhibited a synergistic inhibitory effect on the viability of ovarian cancer cells when combined with DOX. The current study demonstrated that NC inhibited the proliferation and induced the apoptosis of ovarian cancer cells via the Akt signaling pathway and highlighted its potential clinical application for the treatment of ovarian cancer.

Nitidine chloride (NC), an inartificial bioactive alkaloid present in the root of Zanthoxylum nitidum (Roxb.) DC, is known for its versatile anti-inflammation and anticancer capabilities. The molecular mechanisms underlying its anticancer properties, however, remain obscure. The authors of the present study demonstrated the tumor suppressive effects of NC in a human liver cancer cell line using an MTT assay. The tumor suppressive capacity of NC was also analysed in a tumor xenograft nude mouse model. Changes in tumor cell gene expression profiles following NC treatment were detected by microarray; bioinformatics analysis demonstrated that differentially expressed genes were enriched in several cancer-associated pathways, including those initiated by transforming growth factor-β and phosphatidylinositol 4,5-bisphosphate 3-kinase/RAC-α serine/threonine-protein kinase signaling. A Connectivity Map revealed that parthenolide, which has been identified previously as possessing anti-inflammatory and anticancer functions, was potentially extremely similar in molecular function to NC. By screening the data from The Cancer Genome Atlas project, eight genes that were upregulated in liver cancer and significantly suppressed by NC treatment were identified. Overexpression of these genes was closely associated with advanced tumor stage and poor differentiation status. This combination of upregulated genes enabled successful identification and prediction of prognosis for liver cancer. The findings of the present study suggest that NC could inhibit the growth of liver cancer cells through several potential molecular targets and signaling pathways.

The proto-oncogene c-Myc plays critical roles in human malignancies including chronic myeloid leukemia (CML), suggesting that the discovery of specific agents targeting c-Myc would be extremely valuable for CML treatment. Nitidine Chloride (NC), a natural bioactive alkaloid, is suggested to possess anti-tumor effects. However, the function of NC in leukemia and the underlying molecular mechanisms have not been established. In this study, we found that NC induced erythroid differentiation, accompanied by increased expression of erythroid differentiation markers, e. g. α-, ε-, γ-globin, CD235a, CD71 and α-hemoglobin stabilizing protein (AHSP) in CML cells. We also observed that NC induced apoptosis and upregulated cleaved caspase-3 and Parp-1 in K562 cells. These effects were associated with concomitant attenuation of c-Myc. Our study showed that NC treatment in CML cells enhanced phosphorylation of Thr58 residue and subsequently accelerated degradation of c-Myc. A specific group of miRNAs, which had been reported to be activated by c-Myc, mediated biological functions of c-Myc. We found that most of these miRNAs, especially miR-17 and miR-20a showed strong decrement after NC treatment or c-Myc interference. Furthermore, overexpression of c-Myc or miR-17/20a alleviated NC induced differentiation and apoptosis in K562 cells. More importantly, NC enhanced the effects of imatinib in K562 and primary CML cells. We further found that even imatinib resistant CML cell line (K562/G01) and CML primary cells exhibited high sensitivity to NC, which showed potential possibility to overcome imatinib resistance. Taken together, our results clearly suggested that NC promoted erythroid differentiation and apoptosis through c-Myc-miRNAs regulatory axis, providing potential possibility to overcome imatinib resistance.

pDok2, caspase 3 dependent glioma cell growth arrest by nitidine chloride.

Nitidine chloride (NC) possesses anticancer properties in various types of human malignancies. However, the effects of NC on lung cancer cells have not been elucidated. Moreover, the molecular mechanism of NC-involved antitumor activity is unclear. Therefore, we aimed to determine the biological effect of NC and the underlying molecular insights in lung cancer cells. The antineoplastic function of NC was assessed by MTT assays, Annexin V-FITC/PI apoptosis assay, wound healing analysis, and Transwell chamber migration and invasion assay in lung cancer cells. NEDD4 modulation was evaluated by western blotting assays of lung cancer cells after NC treatments. NEDD4 overexpression and downregulation were employed to validate the critical role of NEDD4 in the NC-mediated tumor suppressive effects. We found that NC suppressed cell viability, migration and invasion, but induced apoptosis in lung cancer cells. Mechanistic exploration revealed that NC exhibited its antitumor effects by reducing NEDD4 expression. Furthermore, our rescue experiments dissected that overexpression of NEDD4 abrogated the NC-mediated antineoplastic effects in lung cancer cells. Consistently, downregulation of NEDD4 enhanced the NC-induced anticancer effects. Thus, NC is a promising antitumor agent in lung cancer, indicating that NC might have potential therapeutic applications in the treatment of lung cancer.

Nitidine chloride induces apoptosis and inhibits tumor cell proliferation via suppressing ERK signaling pathway in renal cancer

BACKGROUNDHepatocellular carcinoma (HCC) is at the forefront of the global spectrum of cancer incidence and mortality, with conventional therapies like tyrosine kinase inhibitors limited by resistance. Recent studies have highlighted the promising anticancer effects of nitidine chloride (NC) against HCC. SAC3 domain containing 1 (SAC3D1) is critical for centrosome replication and spindle formation. However, research on SAC3D1 in HCC and NC remains limited.AIMTo investigate the mechanisms underlying SAC3D1’s role in HCC progression and evaluated its potential as a therapeutic target of NC.METHODSRNA sequencing (RNA-seq) identified SAC3D1 expression changes in HCC cells after NC treatment. Molecular docking was further employed to validate the direct binding between NC and SAC3D1. Additionally, HCC multicenter data (The Cancer Genome Atlas_GTEx, ArrayExpress), pathway analysis, Pearson correlation analysis and SAC3D1 in vitro knockdown experiments were integrated to explore the molecular mechanisms underlying SAC3D1's involvement in HCC progression.RESULTSRNA-seq showed that NC treatment significantly downregulated SAC3D1 expression [log2(fold change) = - 0.95, P < 0.05], with molecular docking revealing that NC directly bound to SAC3D1 proteins (binding energy: -9.7 kcal/mol). Enrichment analysis showed that most pathways were closely related to the cell cycle. Pearson correlation analysis indicated that SAC3D1 and cell cycle genes were significantly positively correlated(correlation coefficient ≥ 0.3, P < 0.05). SAC3D1 knockdown inhibited HCC cell invasion, migration, and proliferation by arresting cells in the S and G2/M phases. Flow cytometry confirmed that after SAC3D1 knockdown, the early and total apoptosis percentage of HCC cells decreased, while the late apoptosis percentage increased.CONCLUSIONAs a potential target of NC, SAC3D1 may inhibit HCC progression through cell cycle regulation following its downregulation by NC.

Nitidine chloride (NC), a natural, bioactive, phytochemical alkaloid derived from the roots of Zanthoxylum nitidum, has been reported to exhibit anti-tumor activity against various types of cancer. However, the potential therapeutic role of NC in human cervical cancer has not yet been studied. We are the first to report that NC acts as a potential apoptosis-inducing agent for human cervical cancer in vitro. NC treatment of human cervical cancer cell lines induced caspase-mediated apoptosis, thereby reducing cell viability. Phospho-kinase proteome profiling using a human phospho-kinase array revealed that NC treatment phosphorylated Checkpoint kinase 2 (Chk2) at Thr68, which activates Chk2 in both cell lines. We also found that NC significantly affected the p53/Bim signaling axis, which was accompanied by mitochondrial membrane depolarization and cytochrome c release from the mitochondria into the cytosol. In addition, NC profoundly increased phosphorylation of the histone variant H2AX at Ser139, a typical marker of DNA damage. Taken together, these results provide in vitro evidence that NC can increase Chk2 activation, thereby acting as an attractive cell death inducer for treatment of human cervical cancer.