The anthraquinone derivative KA-4s reduces energy metabolism and enhances the sensitivity of ovarian cancer cells to cisplatin.

Ovarian cancer is the leading cause of death from female gynecological cancers. Cisplatin (DDP) is a first-line drug for ovarian cancer treatment. Due to DDP resistance, there is an urgent need for novel therapeutic drugs with improved antitumor activity. AMPK-mediated metabolic regulatory pathways are related to tumor drug resistance. Our study aimed to determine the relationship between reversing DDP resistance with the anthraquinone derivative KA-4s and regulating AMPK energy metabolism in ovarian cancer. The results showed that KA-4s inhibited the proliferation of ovarian cancer cells. The combination of KA-4s with DDP effectively promoted drug-resistant ovarian cancer cell apoptosis and inhibited cell migration and invasion. Moreover, KA-4s decreased the intracellular ATP level and increased the calcium ion level, leading to AMPK phosphorylation. Further studies suggested that the AMPK signaling pathway may be involved in the mechanism through which KA-4s reduce drug resistance. KA-4s inhibited mitochondrial respiration and glycolysis; downregulated the glucose metabolism-related proteins GLUT1 and GLUT4; the lipid metabolism-related proteins SREBP1 and SCD1; and the drug resistance-related proteins P-gp, MRP1, and LRP. The inhibitory effect of KA-4s on GLUT1 was confirmed by the application of the GLUT1 inhibitor BAY-876. KA-4s combined with DDP significantly increased the expression of p-AMPK and reduced the expression of P-gp. In a xenograft model of ovarian cancer, treatment with KA-4s combined with DDP reduced energy metabolism and drug resistance, inducing tumor apoptosis. Consequently, KA-4s might be evaluated as a new agent for enhancing the chemotherapeutic efficacy of treatment for ovarian cancer.

An autophagy-dependent cell death of MDA-MB-231 cells triggered by a novel Rhein derivative 4F.

Triple negative breast cancer (TNBC) has very poor prognosis and no efficacious therapeutic options due to the absence of a validated molecular target. Therefore, novel therapeutic strategies against TNBC are urgently needed. Our team synthesized and screened a series of compounds derived from Rhein, of which 4F was selected for further analysis based on its ability to produce the vacuolated appearance of cells. Using Cell counting kit-8 assay, colony-formation assay, cell apoptosis and cell cycle assay, we compared the antitumor effects of 4F, Rhein and Cisplatin on a TNBC cell line MDA-MB-231 in vitro. The vacuoles in MDA-MB-231 cells were observed and analyzed by hematoxylin-eosin staining and transmission electron microscopy. Autophagy and apoptosis-related proteins including p62, Microtubule Light Chain 3 (LC3), Beclin-1 and Caspase-3 were determined by western blot. The tandem mRFP-GFP-LC3 Lentivirus was used for monitoring the maturation step of autophagosomes. Our data revealed that 4F had lower cytotoxicity to normal breast cell line MCF-10A as compared with positive drug Doxorubicin. Although 4F had better cytotoxicity than Rhein, it had no influence on cells apoptosis in 4F-treated cells. Accumulation of autolysosomes and autophagosomes was observed in 4F-treated MDA-MB-231 cells, accompanied by increased level of Beclin-1 protein. Enhanced autophagic flux was verified by higher ratio of LC3-II/LC3-I, the degradation of p62 protein and alteration in red and green fluorescence puncta. These findings suggested that the process of MDA-MB-231 cell death induced by 4F seemed rely mainly on autophagy rather than apoptosis. 4F may be an alternative drug candidate against TNBC and merits more exploration.

Inhibition of Anaphase-Promoting Complex by Silence APC/CCdh1 to Enhance Radiosensitivity of Nasopharyngeal Carcinoma Cells.

The aim of this study was to investigate the possibility of APC/CCdh1 as a potential therapeutic target in the radiosensitivity of nasopharyngeal carcinoma (NPC) cell CNE-1, and explain the role of APC subunits after silence of Cdh1 combined with radiotherapy. Transfection with Cdh1 shRNA significantly increased the radiosensitivity of CNE-1 cells and the radiation enhancement ratio (RER) of sh-Cdh1 cells was 1.76. Knockdown of Cdh1 in CNE-1 cells increased irradiation induced apoptosis and G2/M phase cell cycle arrest. The levels of CDC20 and CylinB1 increased and the levels of Ku70 and APC3 decreased after irradiation. APC/CCdh1 is involved in regulation of radiosensitivity in human NPC CNE-1 cells. Our study may provide a promising therapeutic strategy for NPC by targeting Cdh1. J. Cell. Biochem. 118: 3150-3157, 2017. © 2016 Wiley Periodicals, Inc.

Effects of Rubiadin isolated from Prismatomeris connata on anti-hepatitis B virus activity in vitro.

Prismatomeris connata was a kind of Rubiaceae plant for treatment of hepatitis, hepatic fibrosis and silicosis. Whereas, the effective components of Prismatomeris connata remains unexplored. The aim of this study was to investigate the inhibitory effects and mechanisms of Rubiadin isolated from Prismatomeris connata against HBV using HepG2.2.15 cells. The levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay. HBV DNA was qualified q-PCR. Rubiadin was isolated by silica gel column. The structure of the compound was elucidated by HPLC, FT-IR, 1 H-NMR, 13 C-NMR and identified as 1,3-Dihydroxy-2-methyl-9, 10-anthraquinone. Rubiadin significantly decreased HBeAg,HBcAg secretion level and inhibit HBV DNA replication. Rubiadin inhibits the proliferation of the cells and HBx protein expression in a dose-dependent manner. The intracellular calcium concentration was significantly reduced. These results demonstrated that Rubiadin could inhibit HepG2.2.15 cells proliferation, reduce the level of HBx expression, and intracellular free calcium, which might become a novel anti-HBV drug candidate.

CCL18 from tumor-cells promotes epithelial ovarian cancer metastasis via mTOR signaling pathway.

CCL18 is a chemotactic cytokine involved in the pathogenesis and progression of various disorders, including cancer. Previously, our results showed high levels of CCL18 in the serum of epithelial ovarian carcinoma patients suggesting its potential as a circulating biomarker. In this study, we determined that CCL18 expression was up-regulated in ovarian carcinoma compared with adjacent tissue and was expressed in carcinoma cells in the tumor and not in normal ovarian epithelial cells by laser capture microdissection coupled with real-time RT-PCR. Moreover, correlation analysis showed that the CCL18 level was positively correlated with the metastasis of patients with ovarian cancer. Survival analysis also revealed that an increased level of CCL18 was associated with worse survival time in ovarian cancer patients. Over-expression of CCL18 led to enhanced migration and invasion of the Skov3 ovarian cancer cell line in vitro and in vivo. Finally, proteomics analysis demonstrated that CCL18-mediated ovarian cancer invasiveness was strongly correlated with the mTORC2 pathway. These findings suggest that the CCL18 chemokine has an important role in chemokine-mediated tumor metastasis, and may serve as a potential predictor for poor survival outcomes for ovarian cancer. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Effects of a simulated CO2 pneumoperitoneum environment on the proliferation, apoptosis, and metastasis of cervical cancer cells in vitro.

BACKGROUND: This study aimed to investigate the growth curve, cell colony formation, cell cycle, apoptosis, anti-anoikis, and ability of invasion, adhesion, and migration of cervical cancer cells after exposure to a model of a simulated CO2 pneumoperitoneum environment with different pressures and at different times. MATERIAL AND METHODS: The cervical cancer cells were cultured in groups with 8 and 16 mmHg of 100% CO2 for 1, 2, 3, and 4 h in a model of a simulated environment of CO2 pneumoperitoneum. The cells in the control group were cultured in a standard environment. The growth curve was drawn through constant survival cell counts for 7 days, and the group with most obvious change was selected for subsequent experiments to detect cell colony formation, cell cycle apoptosis, and anti-anoikis, and the ability of invasion, adhesion, and migration. RESULTS: After a brief inhibition, the proliferation of cervical cancer cells was markedly increased and had no relationship with different CO2 pressures. Compared with the control group, the early apoptosis rate in the experimental group was higher, and the ability of invasion, migration, and adhesion decreased significantly. CONCLUSIONS: Cervical cancer cells stimulated by a CO2 pneumoperitoneum environment in vitro have an increased the ability to proliferate after a short period of inhibition and have reduced abilities of invasion, migration, and adhesion.

[Characteristics change of the human directional highly lymphatic metastasis ovarian carcinoma cell and venous endothelial cell after establishment of their condition cultrue and co-culture cell system].

OBJECTIVE: To establish the condition cultrue cell system and co- culture cell system with SKOV3/PM4, HUVEC and to study the changes of their biological characteristics. METHODS: The cells of SKOV3/PM4 and HUVEC were labeled with green and red fluorescent respectively. The cell supernatant of SKOV3/PM4 and HUVEC were collected respectively as the condition medium (e.g: the cell supernatant of HUVEC cells was used as SKOV3/PM4 condition medium)and to establish the condition cultrue cell system and the co- culture cell system of the two cell lines. In the condition cultrue cell system, The morphological changes of cells were observed by HE staining to calculate the mitotic index. The ultrastructural changes of the two cells were observed by transmission electron microscopy (TEM). The growth curve of the cells was determined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry was used to analyzed the cell cycles. In the co-culture cell system, the interaction of the two cells were detected by laser scanning confocal microscope (LSCM). The expression of matrix metalloproteinase- 2 (MMP- 2) and matrix metalloproteinase- 9 (MMP- 9) were detected by gelatin zymography. RESULTS: Compared with the single culture SKOV3/PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division, the mitotic index respectively were [(4.8 ± 0.8)%, (11.2 ± 0.3)%; P < 0.05]. The growth rate was significantly increased. In cell cycles, it showed the declined cell ratio of G0/G1 phase, respectively [(69.4 ± 3.6)%, (48.4 ± 4.6)%; P < 0.05] and the raised cell ratio of G2/M phase, respectively [(5.2 ± 1.6)%, (24.9 ± 2.2)%; P < 0.05]. Compared with the single culture HUVEC, the cells which cultured in SKOV3/PM4 condition medium showed the significant morphological change and vacuolization in the cytoplasm, Nuclear division was increased and the mitotic index respectively were [(2.7 ± 0.5)%, (5.7 ± 0.6)%; P < 0.05]. The growth rate was slightly declined. In cell cycles, it showed the raised cell ratio in G0/G1 phase, respectively [(51.4 ± 2.2)%, (79.0 ± 4.1)%; P < 0.05] and the declined cell ratio in G2/M phase, respectively [(19.1 ± 1.2)%, (3.3 ± 0.5)%; P < 0.05]. After co-culture for 48 hours, spontaneous fusion between SKOV3/PM4 and HUVEC cell was observed by the laser confocal microscope. Gelatin zymography assay showed that MMP-2 was not expressed in HUVEC cells, low-expressed in SKOV3/PM4 cells and high-expressed in the co-culture SKOV3/PM4+HUVEC cells. The expression of MMP-2 in co-culture SKOV3/PM4+HUVEC cells and SKOV3/PM4 cells respectively were 1 885 ± 84 and 1 209 ± 114 (P < 0.05). But there were no MMP-9 expression in HUVEC cells, SKOV3/PM4 cells, and the co- culture SKOV3/PM4+HUVEC. CONCLUSION: The characteristics of SKOV3/PM4 and HUVEC show significant changes after condition culture and co-culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway.

Targeted blocking of EGFR and GLUT1 by compound H reveals a new strategy for treatment of triple-negative breast cancer and nasopharyngeal carcinoma.

BACKGROUND: Cytoplasmic epidermal growth factor receptor (EGFR) is overexpressed in both nasopharyngeal carcinoma (NPC) and triple-negative breast cancer (TNBC), while clinical outcome and prognosis vary greatly among patients treated with gefitinib, and all patients eventually develop resistance to this agent. Therefore, we propose a new concept for synthesizing multitarget compounds and reveal new therapeutic strategies for NPC and TNBC expressing EGFR. METHODS: Compound H was synthesized in our previous study. Molecular docking, and cell thermal shift assays (CETSAs) and drug affinity responsive target stability(DARTS) were used to confirm the binding of compound H to EGFR and GLUT1. Methylthiazolyldiphenyl-tetrazolium bromide(MTT), annexin V-PE assays, mitochondrial membrane potential (MMP) assays, and animal models were used to evaluate the inhibitory effect of compound H on TNBC cell lines. Energy metabolism tests, Western blotting, and immunofluorescence staining were performed to evaluate the synergistic effects on EGFR- and glucose transporter type 1(GLUT1)-mediated energy metabolism. RESULTS: Compound H can simultaneously act on the EGFR tyrosine kinase ATP-binding site and inhibit GLUT1-mediated energy metabolism, resulting in reductions in ATP, MMP, intra-cellular lactic acid, and EGFR nuclear transfer. The anti-tumor activity of compound H is significantly superior to the combination of GLUT1 inhibitor BAY876 and EGFR inhibitor gefitinib. Compound H has remarkable anti-proliferative effects on TNBC MDA-MB231 cells, and importantly, no obvious toxicity effects of compound H were found in vivo. CONCLUSIONS: Synergistic effects of inhibition of EGFR- and GLUT1-mediated energy metabolism by compound H may present a new strategy for the treatment of TNBC and NPC.

[Impact of the biological function on epithelial ovarian cancer with ITIH4 gene expression down-regulating in vitro].

OBJECTIVE: To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer. METHODS: The four pairs ITIH4 gene siRNA interference fragments (ITIH4-546, ITIH4-795, ITIH4-917 and ITIH4-1568) were designed respectively, and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently. Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment. The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells. The stably transfected cells- pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418). The experiment was divided into three groups, namely ITIH4-917 transfection group, the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group), and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group). Fluorescence quantitative reverse transcription (RT) PCR and western blot were used to detect the ITIH4 mRNA and protein expression. The cell proliferation, the cell cycle, colony formation of cells, cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT), flow cytometry, colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)], respectively. RESULTS: The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells, the relative copy number was only 0.26 ± 0.15. Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ± 0.10, it significantly lower than that in empty vector group (1.87 ± 0.12, P = 0.008) and negative control group (1.58 ± 0.21, P = 0.032); Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells, empty vector group and negative control group were 0.51, 1.64 and 1.74, respectively, there were statistically significant differences (0.51 vs. 1.64, P = 0.012; 0.51 vs. 1.74, P = 0.014). MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group, and there were statistically significant differences among them (P = 0.001). The S + G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2%, which was significantly higher than that in the empty vector group or negative control group (26.3% and 31.3%, respectively, all P < 0.05). The colony formation rate (55.7 ± 0.7)% in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ± 0.9)% (P = 0.037) and negative control group (31.4 ± 0.3)% (P = 0.043). Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18, whicht was significantly higher than that in the negative control group or empty vector (0.30 ± 0.03, P = 0.031;0.25 ± 0.03, P = 0.028, respectively). Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ± 0.34) was higher than that in the control cells (1.05 ± 0.68) and empty vector group (1.14 ± 0.08), while there were not significant difference (P > 0.05). CONCLUSION: It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.

Novel anthraquinone derivatives trigger endoplasmic reticulum stress response and induce apoptosis.

Background: Endoplasmic reticulum (ER) stress is a therapeutic target in cancer given its regulation of bioenergetics and cell death. Methodology & results: We synthesized 14 ER stress-triggered anthraquinone derivatives by introducing an amino group at the 3-position side chain of the lead compound obtained previously. Most of the anthraquinone derivatives exhibited good antitumor activity due to their ability to induce ER damage through cytoplasmic vacuoles. The mechanisms of ER stress caused by compound KA-4c were related to increasing the expression levels of the ATF6 and Bip proteins and upregulating CHOP and cleaved PARP. Conclusion: Compound KA-4c triggers ER stress response and induces apoptosis via the ATF6-CHOP signaling pathway.

Matrix metalloproteinase-9 expression correlates with prognosis and involved in ovarian cancer cell invasion.

PURPOSE: One of the most important characteristics of ovarian cancer is invasion and metastasis. Matrix metalloproteinases (MMPs) are known to play an important role in cancer cell invasion by mediating the degradation of extracellular matrix (ECM). The activities of MMPs are regulated by tissue inhibitors of metalloproteinases (TIMPs). In this study, we investigated the clinical significance of MMP-2, -7 and -9 and TIMP-1, -2 and -3 expression and MMP-9 functional role in cell invasion and adhesion in ovarian cancer. METHODS: RT-PCR was used to determine mRNA expression of MMP-2, -7 and -9 and TIMP-1, -2 and -3 in ovarian tissues; ELISA was used to detect the serum level of MMP-9; RNA interference (RNAi) was performed to determine the function of MMP-9 in cell invasion and adhesion in ovarian cancer cells. RESULTS: mRNA expression of MMP-2, MMP-7, MMP-9, TIMP-2 and TIMP-3 and serum level of MMP-9 were significantly high in patients with ovarian cancer. MMP-9 expression was significantly high in patients with advanced ovarian cancer and correlated with poor prognosis. The ability of cells for invasion and adhesion was significantly reduced by treatment of cells with MMP-9 siRNA. CONCLUSIONS: Our results suggest that MMP-9 is a potential prognostic factor for ovarian cancer and could be a novel treatment target in ovarian cancer patients.

[Isolation and identification of side population cells in human lung adenocarcinoma cell line A549].

OBJECTIVE: To isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549. METHODS: The protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR. RESULTS: The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells. CONCLUSIONS: SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.

A novel anthraquinone­quinazoline hybrid 7B blocks breast cancer metastasis and EMT via targeting EGFR and Rac1.

At present, effective therapeutic drugs for triple­negative breast cancer (TNBC) are lacking due to the absence of identified or available targets. Therefore, the present study aimed to identify key molecular targets and a specific targeted therapeutic drug to aid with the development of novel therapeutic strategies for TNBC. Based on the high expression of EGFR and Rac1 in TNBC and inspired by a novel antitumor strategy termed combi­targeting, novel anthraquinone­quinazoline hybrid 7B was synthesized to simultaneously target EGFR and Rac1. It was hypothesized that hybrid 7B may possess enhanced potency compared with its parent compounds. Breast cancer cell viability was detected by performing MTT assays. Flow cytometry was conducted to detect the effects of hybrid 7B on the cell cycle, apoptosis and the mitochondrial outer membrane potential. Ultrastructural alterations were observed by transmission electron microscopy. Cell invasion and migration were assessed by performing Transwell and wound­healing assays, respectively. The expression levels of epithelial­mesenchymal transition (EMT) markers and metastasis­related proteins were detected by western blotting. Compared with Rhein and gefitinib, hybrid 7B displayed superior antiproliferative activity in MDA­MB­231 cells with an IC50 value of 2.31 µM, which was 14­fold higher compared with the EGFR tyrosine kinase inhibitor gefitinib. Further experiments demonstrated that hybrid 7B significantly reduced the mitochondrial membrane potential, enhanced MDA­MB­231 cell apoptosis and induced cell cycle arrest at the G2/M phase compared with the control group. Typical morphological alterations of apoptotic cells were observed in hybrid 7B­treated MDA­MB­231 and MCF­7 cells. Compared with the control group, hybrid 7B significantly inhibited MDA­MB­231 cell invasion and migration by downregulating Rac1, EGFR, matrix metalloproteinases, snail family transcriptional repressor 1, Vimentin and ß­catenin protein expression levels, and upregulating E­cadherin protein expression levels. The present study demonstrated that hybrid 7B inhibited TNBC cell migration and invasion by reversing EMT and targeting EGFR and Rac1; therefore, hybrid 7B may serve as a promising therapeutic agent for TNBC.

[Expression and significance of differentially expressed protein endoplasmic reticulum aminopeptidase 1 in ovarian carcinoma with lymph node metastasis].

OBJECTIVE: To detect the genetic transcription and protein expression level of endoplasmic reticulum aminopeptidase 1 (ERAP1) in human ovarian cancer cells and tissue and study their relationship with directional lymphatic metastasis. METHODS: Real-time fluorescent quantitative PCR and western blot were used to determine the expressions of ERAP1 gene and protein in the human ovarian cancer cell lines between non-directional (SKOV3) and directional highly lymphatic metastasis (SKOV3-pm2, SKOV3-pm3, SKOV3-pm4). Immunohistochemistry method was used to further validate the ERAP1 expressions of the transplanted ovarian tumor primarily focus and the lesions of lymph node metastasis from nude mice and the human ovarian cancer primarily focus and the lesions of lymph node metastasis. RESULTS: The expression level of ERAP1 gene and protein were down-regulated in SKOV3, SKOV3-pm2, SKOV3-pm3, SKOV3-pm4 cell sublines (0.118 ± 0.012, 0.031 ± 0.003, 0.028 ± 0.003, 0.016 ± 0.005; 0.91 ± 0.33, 0.09 ± 0.03, 0.10 ± 0.04, 0.05 ± 0.04; respectively), and the level of ERAP1 in SKOV3 cell was higher than those in the other three kinds of cell lines (P < 0.05). The results showed that there were significant declining trend of expression of ERAP1 in the human ovarian cancer cell lines between non-directional and directional highly lymphatic metastasis; the transplanted ovarian tumor primarily focus and the metastasis lesions of lymph node from nude mice (143 ± 22 vs. 97 ± 12, P < 0.05), the primarily focus (184 ± 14) and the lesions of lymph node metastasis from human ovarian tumors (P < 0.05). CONCLUSION: The absence or down-regulated expression of ERAP1 is closely related to the metastasis and invasion of lymph node in ovarian carcinoma.

[Relationship between cathepsin L and invasion and metastasis of ovarian carcinoma cells].

OBJECTIVE: To clone cathepsin L (CTSL) gene and construct the eukaryotic expression plasmid pcDNA3.1-CTSL and study the relationship between CTSL and invasion and metastasis in ovarian cancer cells in vitro. METHODS: The total RNA was extracted from the ovarian cancer tissue and the intact cDNA of CTSL was applied by reverse transcription (RT)-PCR. The product of RT-PCR was cloned to pMD18-T vector, and subcloned to pcDNA3.1 vector. It was tested by the enzymation and DNA sequencing. The eukaryotic expression plasmid of CTSL was introduced into HO8910 cells by liposome transfection reagent. RT-PCR was used to confirm the recombinant plasmid DNA integrated with the genomic DNA of HO8910 cells. Western blot was used to confirm the CTSL protein expression in positive clones cells. The cell growth curves, clonogenicity efficiency were observed. The cell cycles were measured by flow cytometer. The ability of invasion, metastasis and adhesion of ovarian cancer cells were detected by the matrigel invasion assay, transwell migration assay and adhesion assay, respectively. RESULTS: The results from restrictive enzyme analysis and sequencing showed that the CTSL gene was successfully inserted into pcDNA3.1. Result from RT-PCR and western blot showed that the ovarian cancer cells which transfected by recombinant plasmid could express CTSL gene and protein. There was no difference between HO8910-CTSL and HO8910-pcDNA3.1 cells in proliferation and adhesion ability (0.16 ± 0.04 versus 0.19 ± 0.04) of the cells (P > 0.05). There was difference between HO8910-CTSL and HO8910-pcDNA3.1 cells in matrigel invasion ability (0.34 ± 0.18 versus 0.17 ± 0.04) and metastasis ability (1.252 ± 0.114 versus 0.486 ± 0.027) of cancer (all P < 0.05). CONCLUSION: CTSL maybe increase the ability of invasion and metastasis of ovarian cancer cells in vitro, which may be a molecular target of blocking invasion and metastasis of ovarian cancer.

Novel anthraquinone compounds as anticancer agents and their potential mechanism.

Anthraquinones exhibit a unique anticancer activity. Since their discovery, medicinal chemists have made several structural modifications, resulting in the design and synthesis of a large number of novel anthraquinone compounds with different biological activities. In general, anthraquinone compounds have been considered to have anticancer activity mainly through DNA damage, cycle arrest and apoptosis. However, recent studies have shown that novel anthraquinone compounds may also inhibit cancer through paraptosis, autophagy, radiosensitising, overcoming chemoresistance and other methods. This Review article provides an overview of novel anthraquinone compounds that have been developed as anticancer agents in recent years and focuses on their anticancer mechanism.

Rhein Derivative 4F Inhibits the Malignant Phenotype of Breast Cancer by Downregulating Rac1 Protein.

BACKGROUND: Triple-negative breast cancer is a common malignant tumor with unfavorable prognosis affecting women worldwide; thus, there is an urgent need for novel therapeutic drugs with improved anti-tumor activity. Rac family small GTPase 1 (Rac1) plays an important role in malignant behavior and is a promising therapeutic target. We reported an anthraquinone compound, Rhein, and its derivative, 4F, and investigated their downregulation effects on Rac1 in breast cancer cells in vitro. METHODS: The inhibition of cell proliferation by derivative 4F was investigated in two breast cancer (MDA-MB-231 and MCF-7) and normal breast (MCF-10A) cell lines by cell counting kit-8 assay and growth curves. The role of 4F in cell migration and invasion and cytoskeletal change were assessed by Transwell chamber assay and F-actin staining, respectively. The affinity of Rhein and its derivative for Rac1 protein and the regulation of Rac1 promoter activity were evaluated by molecular docking software and luciferase reporter gene assay, respectively. Rac1 protein expression was determined by western blot assay. RESULTS: Compared to Rhein, derivative 4F more strongly inhibited breast cancer cell proliferation, migration, and invasion and also cause cytoskeletal changes like those in paclitaxel. Derivative 4F not only bound more stably to Rac1 but also inhibited Rac1 promoter activity in cells and downregulated Rac1 protein expression. CONCLUSIONS: Rhein derivative 4F is a new anthraquinone compound with better anti-tumor activity than that of the lead compound Rhein in breast cancer. It down-regulated Rac1 expression and may be a small molecule inhibitor of Rac1.

Rac1: A potential radiosensitization target of human nasopharyngeal carcinoma CNE2 cells.

Radiotherapy has a high cure rate for early nasopharyngeal carcinoma(NPC). However, the radiation resistance of poorly differentiated NPC cells impacts the effectiveness of treatment of early-stage NPC patients. Here, we explored the relationship between Ras-related C3 botulinum toxin substrate 1(Rac1) expression and NPC radiosensitivity. In vitro and in vivo studies revealed that upregulation of Rac1, when combined with X-ray treatment, increased growth inhibition and induced remarkable morphological changes and apoptosis in CNE2 cells. Furthermore, rupturing of the cell and nuclear membranes, degeneration of the cristae and significant swelling of the mitochondria were observed, which were consistent with the high apoptotic rate. The Rac1(+) cells exhibited approximately 50% more migration compared with that of the NC and Rac1(-) cells. The overexpression of Rac1 can increase the radiation sensitivity of NPC CNE2 cells, and the mechanism may be closely related to the oxidative damage of mitochondria. Rac1 might be a potential target for radiosensitization in poorly differentiated NPC.

Synthesis and screening of novel anthraquinone-quinazoline multitarget hybrids as promising anticancer candidates.

Aim: The EGF receptor (EGFR) is overexpressed in multiple epithelial-derived cancers and is considered to be a vital target closely associated with cancer therapy. In this study, a series of novel anthraquinone-quinazoline hybrids targeting several vital sites for cancer therapy were designed and synthesized. Methodology & results: Most of the synthesized hybrids demonstrated excellent antiproliferative activity and downregulation of the expression of EGFR. The most promising compound 7d showed the strongest antiproliferation activity; this compound significantly downregulated the expression of p-EGFR protein, induced a remarkable apoptosis effect, promoted the rearrangement of F-actin filaments and destruction of cytoskeleton, induced DNA damage and enhanced radiosensitivity of A549 cells. Conclusion: The novel anthraquinone-quinazoline hybrid 7d emerges as an anticancer drug candidate with promising multitargeted biological activities.

Confirming whether novel rhein derivative 4a induces paraptosis-like cell death by endoplasmic reticulum stress in ovarian cancer cells.

Ovarian cancer is the leading cause of death among gynecologic cancer patients. Although platinum-based chemotherapy as a frontline treatment for ovarian cancer has been widely used in clinical settings, its clinical efficacy is not satisfactory due to the resistance of ovarian cancer cells to apoptosis. Therefore, it is of great significance to induce non-apoptotic programed cell death patterns, such as paraptosis, in ovarian cancer. In this study, we aimed to explore the potential anticancer mechanisms of novel rhein derivative 4a, which was modified with rhein as a lead compound. The results showed that a wide range of vacuoles from the endoplasmic reticulum and mitochondria appeared in ovarian SKOV3, SKOV3-PM4, and A2780 cells treated with derivative 4a, and the cell death caused by derivative 4a is a type of non-apoptotic and non-autophagic death, which is caused by expansion and damage of the endoplasmic reticulum or mitochondria, showing the characteristics of para-apoptotic death. Furthermore, derivative 4a stimulated the unfolded protein reaction of ovarian cancer cells by upregulating the expression of Bip78 and activating the PERK-eIF2α-ATF4 pathways. Notably, rhein derivative 4a-induced cell death was positively correlated with activation of p38, ERK, and JNK, and negatively correlated with Alix, a known protein that inhibits paraptosis. In addition, derivative 4a treatment also induced G2/M phase arrest in ovarian cancer cells. Taken together, our study reveals that derivative 4a induces paraptosis, and this finding can serve as a basis in developing a new strategy for the treatment of antiapoptotic ovarian cancer.