Folic acid modified TPGS as a novel nano-micelle for delivery of nitidine chloride to improve apoptosis induction in Huh7 human hepatocellular\u2002carcinoma

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) has been demonstrated to exert a tumor-suppressive function in various types of human cancers. However, the detailed mechanism of NC-mediated anti-tumor effects remains elusive. It has been reported that SIN1, a component of mTORC2 (mammalian target of rapamycin complex C2), plays an oncogenic role in a variety of human cancers. Therefore, the inhibition of SIN1 could be useful for the treatment of human cancers. In this study, we explored whether NC triggered an anti-cancer function via the inhibition of SIN1 in osteosarcoma (OS) cells. An MTT assay was performed to measure the effect of NC on the cell growth of osteosarcoma cells, and flow cytometry was used to detect the apoptotic rate of the cells after NC treatment. The expression of SIN1 was detected by western blotting. Wound-healing assay and Transwell chamber invasion assay were conducted to analyze the motility of osteosarcoma cells following NC exposure. We found that exposure to NC led to the inhibition of cell growth, migration, and invasion and the induction of apoptosis. Mechanistically, we found that NC inhibited the expression of SIN1 in osteosarcoma cells. Overexpression of SIN1 abrogated the inhibition of cell growth and motility induced by NC in osteosarcoma cells. Our results indicate that NC exhibits its tumor-suppressive activity via the inhibition of SIN1 in osteosarcoma cells, suggesting that NC could be a potential inhibitor of SIN1 in osteosarcoma.

BackgroundStemness of CD133+EPCAM+ hepatocellular carcinoma cells ensures cancer resistance to apoptosis,which is a challenge to current liver cancer treatments. In this study, we evaluated the tumorcidal activity of a novel nanoparticle of nitidine chloride (TPGS-FA/NC, TPGS-FA: folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate, NC: nitidine chloride), against human hepatocellular carcinoma (HCC) cell line Huh7 growth in vitro and in vivo.MethodsHuh7 cells were treated with TPGS-FA/NC. Cell proliferation was assessed using MTT and colony assays. The expression of cell markers and signaling proteins was detected using western blot analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Huh7 cells. TPGS-FA/NC (7.5, 15, 30, 60, 120 μg/mL) dose-dependently inhibited the proliferation of HCC cells, which associated with a reduction in AQP3 and STAT3 expression. Importantly,TPGS-FA/NC (10, 20, and 40 μg/mL) significantly reduced the EpCAM+/CD133+cell numbers, suppressed the sphere formation. The in vivo antitumor efficacy of TPGS-FA/NC was proved in Huh7 cell xenograft model in BALB/c nude mice, which were administered TPGS-FA/NC(4 mg· kg − 1· d − 1, ig) for 2 weeks.ResultsTPGS-FA/NC dose-dependently suppressed the AQP3/STAT3/CD133 axis in Huh7 cells. In Huh7 xenograft bearing nude mice, TPGS-FA/NC administration markedly inhibited Huh7 xenograft tumor growth .ConclusionsTPGS-FA/NC inhibit HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the clinical therapy of hepatocellular carcinoma.

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.

Nitidine chloride (NC) has displayed anti-tumor effects in various types of cancer. However, the role of NC in human melanoma is largely unknown. This study aimed to investigate the effects of NC on melanoma cancer cells A375 and WM35 by MTT assay. Apoptosis was measured by detecting caspase-3 protein expression level and its activity. Autophagy was measured by TEM image, immunostaining and immunoblotting assays. MTT assays showed that NC significantly blocks melanoma cells proliferation. Immunoblotting and caspase-3 activity assays showed that NC inhibited melanoma cells proliferation by inducing cell apoptosis. TEM, immunostaining and immunoblotting assays showed that NC also triggers melanoma cells autophagy and activation of the AMPK-mTOR pathway, which plays an important role in autophagy initiation. Finally, we found that blocking autophagy by 3-MA or AMPK pathway inhibitor greatly enhanced NC-induced apoptosis and cell death, indicating that NC-induced autophagy may have a cytoprotective effect in melanoma cells. Together, these results suggested that NC has strong anti-tumor effects on melanoma cells.

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

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.

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) 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.

BackgroundThe development of hepatic cancer is tightly regulated by multiple intracellular signaling pathways. Therefore, most currently-used anti-tumor agents, which typically target single intracellular pathway, might not always be therapeutically effective. Additionally, long-term use of these agents probably generates drug resistance and unacceptable adverse effects. These problems increase the necessity for the development of new chemotherapeutic approaches. Nitidine chloride (NC), a natural benzophenanthridine alkaloid, has been shown to inhibit cancer growth via induction of cell apoptosis and suppression of cancer angiogenesis. But the precise mechanisms of its tumorcidal activity are not well understood.MethodsTo further elucidate the precise mechanisms of its anti-tumor activity, using a hepatic cancer mouse xenograft model, the human hepatic cancer cell lines (HepG2, HCCLM3, Huh7), and umbilical vein endothelial cells (HUVEC), here we evaluate the effect of NC on tumor growth in vivo and in vitro and investigated the underlying molecular mechanisms.ResultsWe found that NC treatment resulted in significant decrease in tumor volume and tumor weight respectively, but didn’t affect body weight changes. Additionally, NC treatment dose- and time-dependently reduced the cell viability of all three hepatic cell lines. Moreover, NC suppressed the activation of STAT3, ERK and SHH pathways; and altered the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4, VEGF-A and VEGFR2. These molecular effects resulted in the promotion of apoptosis, inhibition of cell proliferation and tumor angiogenesis.ConclusionsOur findings suggest that NC possesses a broad range of anti-cancer activities due to its ability to affect multiple intracellular targets, suggesting that NC could be a novel multi-potent therapeutic agent for the treatment of hepatic cancer and other cancers.

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.

Nitidine chloride (NC) has been demonstrated to exert anti-tumor effects on various types of tumor. However, no studies have investigated the anti‑metastatic effect of NC on ovarian cancer cells, and the underlying mechanisms have not yet been clearly established. The present study aimed to determine the effect of NC on the migration and invasion of ovarian cancer cells. Cell viability and proliferation of ovarian cancer cells were assessed by MTT assay. A scratch wound healing assay and Transwell assays were performed to detect migration and invasion of cells, respectively. The expression levels of matrix metalloproteinase (MMP)‑2 and 9 were detected at the mRNA and protein level following stimulation with NC. Subsequently, the expression of mitogen‑activated protein kinases was detected by western blot analysis. Finally, an inhibitor of extracellular signal‑regulated kinase (ERK) was applied to investigate the effect of NC on the expression of MMP‑2/9 as well as the migration and invasion of cells. It was found that NC suppressed the proliferation, migration and invasion of A2780 ovarian cancer cells. NC downregulated MMP‑2 and MMP‑9 in a dose‑ and time‑dependent manner. In addition, NC was also able to downregulate phosphorylation of ERK. Furthermore, by applying an ERK inhibitor, U0126, the effect of NC on the expression of MMP-2/9 and inhibition of cell migration and invasion was verified. Taken together, these results demonstrated that NC inhibited the migration and invasion of ovarian cancer cells via the ERK signaling pathway.

BackgroundAs the increasing mortality and incidence of lung cancer (LC), there is an urgent need to discover novel treatment agent. In this study, we aimed to investigate the anti-LC effects of nitidine chloride (NC), a small molecular compound extracted from Chinese herbal medicine, while detailing its underlying mechanisms.MethodsCell viability was detected by MTT assays and five cell death inhibitors, including ferrostatin-1 (Fer-1), Z-VAD-FMK, necrostatin-1 (Nec-1), disulfiram (DSF) and IM-54 were used to explore the type of cell death induced by NC. The microscopic features of NC-induced pyroptosis were assessed by transmission electron microscopy (TEM) and the pyroptotic-related proteins such as caspase and gasdermin family, were examined by western blot. Network pharmacology was employed to predict the potential mechanisms of NC in lung cancer treatment. CETSA and DARTs were used to determine the activity of NC binding to targeted protein. Xenograft mice model was established to further investigate the inhibitory effect and mechanism of NC against LC.ResultsThe pyroptosis inhibitor (DSF) and apoptosis inhibitor (Z-VAD-FMK) but not IM-54, necrostatin-1, or Ferrostatin-1 rescued NC-induced cell death. Morphologically, H1688 and A549 cells treated with NC showed notably pyroptotic features, such as cell swelling and large bubbles emerging from the plasma membrane. Gasdermin E (GSDME) rather than GSDMC or GSDMD was cleaved in NC-treated H1688 and A549 cells with an increased cleavage of caspase 3. Combined with network pharmacology and molecule docking, PI3K/Akt signaling axis was predicted and was further verified by CETSA and DARTs assay. In addition, the activation of PI3K is able to rescue the pyroptosis induced by NC in vitro. In xenograft model of LC, NC significantly hindered the transduction of PI3K-AKT pathway, inducing pyroptosis of tumor.ConclusionOur data indicated that NC is a potential therapeutic agent for the treatment of LC via triggering GSDME-dependent pyroptosis.

Signal transducer and activator of transcription 3 (STAT3) is persistently activated in cancer cells and contributes to malignant progression in various types of cancer. The Janus-activated kinase (JAK) family phosphorylates STAT3 in response to stimulation by cytokines or growth factors. The JAK1-STAT3 signaling pathway plays an important role in cell proliferation and apoptosis. Nitidine chloride (NC) is a benzophenanthridine alkaloid that has been reported as an antitumor agent due to its its inhibitory effects on topoisomerase I. Using a mouse xenograft model of hepatocellular carcinoma (HCC), this study aimed to evaluate the effects of NC on tumor growth in vivo and to elucidate the underlying mechanisms. The analysis of the effects of NC on apoptosis in HCC tumor xenografts in mice was carried out by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay; the expression of Bcl-2, Bax, cyclin-dependent kinase (CDK)4, cyclin D1, p21 and proliferating cell nuclear antigen (PCNA) was analyzed by immunohistochemistry; and the protein expression of JAK1 and STAT3 was examined by western blot analysis. Our results revealed that treatment with NC decreased the tumor volume and tumor weight, suggesting that NC inhibits HCC cell growth in vivo. In addition, NC blocked the activation of JAK1-STAT3 in the tumor tissues, which in turn resulted in the induction of cancer cell apoptosis and the inhibition of proliferation. Consequently, treatment with NC downregulated the expression of cyclin D1, CDK4 and Bcl-2 and increased the level of p21 and Bax. Our data provide a molecular basis for the antitumor activity of NC.

Nature is an abundant and largely untapped source of potent bioactive molecules. Ribosome biogenesis modulators have proven effective in suppressing cancer cell growth and are currently being evaluated in clinical trials for anticancer therapies. In this study, we characterized the alkaloid nitidine chloride (NC), produced by the endemic Cameroonian plant Fagara (and other plants). We demonstrate that NC kills cancer cells regardless of their p53 status and inhibits tumor growth in vitro. Furthermore, NC profoundly suppresses global protein synthesis. Treatment of human cells with NC causes severe nucleolar disruption and inhibits pre-rRNA synthesis by destabilizing key factors required for recruitment of RNA polymerase I to ribosomal DNA promoters. In vitro, NC intercalates into DNA and inhibits topoisomerases I and II. Consistently, NC treatment activates a DNA damage response. We propose that the torsional stress on rDNA caused by topoisomerase inhibition leads to loss of RNA polymerase I function and to shutdown of ribosome biogenesis. Although NC has long been suspected of possessing anticancer properties, here we provide a molecular explanation for its mechanism of action. In budding yeast cells, interestingly, NC inhibits cell growth, impairs ribosome biogenesis, and disrupts nucleolar structure. This suggests that its mode of action is at least partially evolutionarily conserved.