IMB5036, a novel pyridazinone compound, inhibits hepatocellular carcinoma growth and metastasis.

Background Hepatocellular carcinoma (HCC) is one of the most common cancers with a high mortality rate due to metastasis and relapse. Purpose Here, we reported a small-molecule pyridazinone compound, designated as IMB5036. Its antitumor activity against HCC and underlying mechanism were studied. Methods In vitro cytotoxicity, apoptosis, DNA breaks, and cell motility assays were performed. Protein expression was analyzed by Western blot and microarray analysis. A xenograft tumor model in athymic mice was used to evaluate the antitumor activity. Results IMB5036 displayed potent cytotoxicity against various HCC cell lines. It caused double DNA breakages and induced cell death via apoptosis. It also significantly inhibited the motility of HCC cells. Western blot showed that IMB5036 induced the up-regulation of E-cadherin, while down-regulation of N-cadherin. The gene expression profile analysis and Western blot assay revealed that IMB5036 down-regulated the expression of Tau protein. Analysis of the TCGA dataset revealed that high expression of Tau decreased the survival rate of HCC patients. In vivo experiments proved that IMB5036 significantly inhibited the growth of HCC xenografts in athymic mice. Conclusions These results collectively demonstrate IMB5036 can be a promising therapeutic candidate for patients with HCC.

[Programmed Necrosis Inducers for Cancer Treatment].

Programmed necrosis,a mode of cell death independent of Caspase,is mainly mediated by receptor-interacting protein kinase-1 (RIPK1),receptor-interacting protein kinase-3 (RIPK3),and mixed lineage kinase domain-like protein (MLKL).Studies have demonstrated that programmed necrosis has the dual role of promoting and inhibiting tumor growth and thus we can control the development of tumor by regulating programmed necrosis.The drugs capable of inducing programmed necrosis show potential anti-tumor activity.In addition,inducing programmed necrosis is an effective way to overcome tumor resistance to apoptosis.This paper summarized the mechanisms of programmed necrosis and its relationship with tumors.We focused on the antitumor activity of programmed necrosis inducers including natural products,chemotherapeutic drugs,death receptor ligands,kinase inhibitors,inorganic salts,metal complexes,and metal nanoparticles.These agents will provide new therapeutic candidates for the treatment of tumors,especially the tumors acquiring resistance to apoptosis.

Focal adhesion kinase is activated by microtubule-depolymerizing agents and regulates membrane blebbing in human endothelial cells.

Microtubule-depolymerizing agents can selectively disrupt tumor vessels via inducing endothelial membrane blebbing. However, the mechanism regulating blebbing is largely unknown. IMB5046 is a newly discovered microtubule-depolymerizing agent. Here, the functions of focal adhesion kinase (FAK) during IMB5046-induced blebbing and the relevant mechanism are studied. We found that IMB5046 induced membrane blebbing and reassembly of focal adhesions in human vascular endothelial cells. Both FAK inhibitor and knock-down expression of FAK inhibited IMB5046-induced blebbing. Mechanism study revealed that IMB5046 induced the activation of FAK via GEF-H1/ Rho/ ROCK/ MLC2 pathway. cRGD peptide, a ligand of integrin, also blocked IMB5046-induced blebbing. After activation, FAK further promoted the phosphorylation of MLC2. This positive feedback loop caused more intensive actomyosin contraction and continuous membrane blebbing. FAK inhibitor blocked membrane blebbing via inhibiting actomyosin contraction, and stimulated stress fibre formation via promoting the phosphorylation of HSP27. Conclusively, these results demonstrate that FAK is a molecular switch controlling endothelial blebbing and stress fibre formation. Our study provides a new molecular mechanism for microtubule-depolymerizing agents to be used as vascular disrupting agents.

LINC00467 promotes cell proliferation and metastasis by binding with IGF2BP3 to enhance the mRNA stability of TRAF5 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has been recognized as a member of the most common human malignant tumors globally. According to multiple studies, long noncoding RNAs (lncRNAs) have been defined as vital regulators in tumor progression. Although previous studies have indicated that lncRNA long intergenic non-protein coding RNA 467 (LINC00467) exerts oncogenic effect in tumorigenesis and the development of cancers, the specific function that LINC00467 induces in HCC remains obscure. METHODS: LINC00467 expression was examined by a quantitative reverse transcriptase-polymerase chain reaction. CCK-8, EdU, transwell, western blotting and caspase-3 activity analyses were utilized to testify the role of LINC00467 in HCC. The interaction between IGF2BP3 and LINC00467 (or TRAF5) was investigated by luciferase reporter, RIP and RNA pull-down assays. RESULTS: LINC00467 upregulation in HCC tissues and cells was observed. LINC00467 silencing suppressed cell proliferation and metastasis, whereas it facilitated cell apoptosis in HCC. The gene for tumor necrosis factor receptor-associated factor 5 (TRAF5) was a neighboring gene of that for LINC00467 and its expression was positively modulated by LINC00467 in HCC. TRAF5 knockdown inhibited HCC progression. LINC00467 deficiency could decrease the mRNA stability of TRAF5 in HCC. Insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) could bind with LINC00467 and its depletion could lower TRAF5 mRNA stability in HCC. Final rescue assays further indicated that downregulation of IGF2BP3 or TRAF5 acted against LINC00467 upregulation-mediated function on HCC progression. CONCLUSIONS: LINC00467 promotes cell proliferation and metastasis by binding with IGF2BP3 to enhance the mRNA stability of TRAF5 in HCC.

Development of a prognostic score for recommended TACE candidates with hepatocellular carcinoma: A multicentre observational study.

BACKGROUND & AIMS: Previous prognostic scores for transarterial chemoembolization (TACE) were mainly derived from real-world settings, which are beyond guideline recommendations. A robust model for outcome prediction and risk stratification of recommended TACE candidates is lacking. We aimed to develop an easy-to-use tool specifically for these patients. METHODS: Between January 2010 and May 2016, 1,604 treatment-naïve patients with unresectable hepatocellular carcinoma (HCC), Child-Pugh A5-B7 and performance status 0 undergoing TACE were included from 24 tertiary centres. Patients were randomly divided into training (n = 807) and validation (n = 797) cohorts. A prognostic model was developed and subsequently validated. Predictive performance and discrimination were further evaluated and compared with other prognostic models. RESULTS: The final presentation of the model was "linear predictor = largest tumour diameter (cm) + tumour number", which consistently outperformed other currently available models in both training and validation datasets as well as in different subgroups. The thirtieth percentile and the third quartile of the linear predictor, namely 6 and 12, were further selected as cut-off values, leading to the "six-and-twelve" score which could divide patients into 3 strata with the sum of tumour size and number ≤6, >6 but ≤12, and >12 presenting significantly different median survival of 49.1 (95% CI 43.7-59.4) months, 32.0 (95% CI 29.9-37.5) months, and 15.8 (95% CI 14.1-17.7) months, respectively. CONCLUSIONS: The six-and-twelve score may prove an easy-to-use tool to stratify recommended TACE candidates (Barcelona Clinic Liver Cancer stage-A/B) and predict individual survival with favourable performance and discrimination. Moreover, the score could stratify these patients in clinical practice as well as help design clinical trials with comparable criteria involving these patients. Further external validation of the score is required. LAY SUMMARY: There is currently no prognostic model specifically developed for recommended or ideal transarterial chemoembolization (TACE) candidates with hepatocellular carcinoma, despite these patients being frequently identified as the best target population in pivotal randomized controlled trials. The six-and-twelve score provides patient survival prediction, especially in ideal candidates of TACE, outperforming other currently available models in both training and validation sets, as well as different subgroups. With cut-off values of 6 and 12, the score can stratify ideal TACE candidates into 3 strata with significantly different outcomes and may shed light on risk stratification of these patients in clinical practice as well as in clinical trials.

Deletion of INMAP postpones mitotic exit and induces apoptosis by disabling the formation of mitotic spindle.

INMAP was first identified as a spindle protein that plays important roles in cell-cycle progression, and previous studies have revealed that its abnormal expression leads to mitotic disorder and the growth inhibition of human tumor xenografts, but the underlying mechanism is still unclear. In this study, we knocked out INMAP in HEK293T cells, a strain of human embryonic renal cells, through CRISPR-Cas9 gene editing technology, resulting in obvious cell growth inhibition. In this system, the deletion of INMAP caused obviously apoptosis. And we also found that knockout of INMAP caused micronuclei formation, chromosome aberration, and γH2AX expression upregulation, suggesting DNA damage induction and genomic stability impairment. As a principal component of spindle, the expression of ß-tubulin, detected through Western blot, is obviously upregulated in HEK293T-INMAP-/-. Meanwhile, the level of Cyclin B is also upregulated, whereas, that of Cyclin E, downregulated, with the postponement of mitotic exit and the assembly anomaly of spindle. These results suggest that the deletion of INMAP block the formation of spindle, leading to arrest of cell cycle and DNA damage, finally blocking cell proliferation and inducing apoptosis. Therefore, INMAP is an indispensable factor for genomic integrity and normal mitotic exit.

The actomyosin network is influenced by NMHC IIA and regulated by CrpF46, which is involved in controlling cell migration.

When a cell migrates, the centrosome positions between the nucleus and the leading edge of migration via the microtubule system. The protein CrpF46 (centrosome-related protein F46) has a known role during mitosis and centrosome duplication. However, how CrpF46 efficiently regulates centrosome-related cell migration is unclear. Here, we report that knockdown of CrpF46 resulted in the disruption of microtubule arrangement, with impaired centrosomal reorientation, and slowed down cell migration. In cells that express low levels of CrpF46, stress fibers were weakened, which could be rescued by recovering Flag-CrpF46. We also found that CrpF46 interacted with non-muscle myosin high chain IIA (NMHC IIA) and that its three coiled-coil domains are pivotal for its binding to NMHC IIA. Additionally, analyses of phosphorylation of NMHC IIA and RLC (regulatory light chain) demonstrated that CrpF46 was associated with myosin IIA during filament formation. Indirect immunofluorescence images indicated that NM IIA filaments were inhibited when CrpF46 was under-expressed. Thus, CrpF46 regulates cell migration by centrosomal reorientation and altering the function of the actomyosin network by controlling specific phosphorylation of myosin.

A CD13-targeting peptide integrated protein inhibits human liver cancer growth by killing cancer stem cells and suppressing angiogenesis.

CD13 is a marker of angiogenic endothelial cells, and recently it is proved to be a biomarker of human liver cancer stem cells (CSCs). Herein, the therapeutic effects of NGR-LDP-AE, a fusion protein composed of CD13-targeting peptide NGR and antitumor antibiotic lidamycin, on human liver cancer and its mechanism were studied. Western blot and immunofluorescence assay demonstrated that CD13 (WM15 epitope) was expressed in both human liver cancer cell lines and vascular endothelial cells, while absent in normal liver cells. MTT assay showed that NGR-LDP-AE displayed potent cytotoxicity to cultured tumor cell lines with IC50 values at low nanomolar level. NGR-LDP-AE inhibited tumorsphere formation of liver cancer cells, and the IC50 values were much lower than that in MTT assay, indicating selectively killing of CSCs. In endothelial tube formation assay, NGR-LDP-AE at low cytotoxic dose significantly inhibited the formation of intact tube networks. Animal experiment demonstrated that NGR-LDP-AE inhibited the growth of human liver cancer xenograft. Immunohistochemical analysis showed that NGR-LDP-AE induced the down-regulation of CD13. In vitro experiment using cultured tumor cells also confirmed this result. NGR-LDP-AE activated both apoptotic and autophagic pathways in cultured tumor cells, while the induced autophagy protected cells from death. Conclusively, NGR-LDP-AE exerts its antitumor activity via killing liver CSCs and inhibiting angiogenesis. With one targeting motif, NGR-LDP-AE acts on both liver CSCs and angiogenic endothelial cells. It is a promising dual targeting fusion protein for liver cancer therapy, especially for advanced or relapsed cancers.

[Isolation and culture of mouse spermatogonial stem cells and determination of the related markers].

OBJECTIVE: To establish a simple and highly effective isolation and culture system of mouse spermatogonial stem cells(SSCs)and detect the expression of stem cell-related markers in the isolated cells. METHODS: The structures of seminiferous tubules of neonatal(6-8 days of age)and adult(26-28 weeks)DBA/2 mice were compared using histochemical examination. Testes of neonatal mice were selected for preparing primary cells. The digestive efficiency of different enzymes was compared. SSCs were isolated according to the different binding abilities of testicle somatic cells and SSCs to gelatin matrix. The effects of different base culture media such as StemPro34 and α-MEM,gelatin,and serum on the SSCs binding activity and growth were studied. The cell morphology was observed during the culture process. Immunofluorescence was used to detect the expression of SSCs and cancer stem cells(CSCs)-related markers in SSCs. RESULTS: The content of SSCs in the testes of neonatal mice was relatively higher than that in adult mice. Trypsin showed the highest digestive efficiency. In StemPro34 supplemented with 1% fetal bovine serum and on the gelatin matrix,testicular somatic cells could bind with the plate efficiently. Spermatogonial cells grew well when using mitomycin C-treated testicular somatic cells as feeder cells and showed typical characteristic of SSCs. After 13 days of culture,spermatogonial cells formed cell clusters. Immunofluorescence assay showed that SSCs markers glial cell line-derived neurotrophic factor(GDNF)family receptor α1(GFRα1)and VASA protein were highly expressed in the cell clusters. CSCs marker CD44 was expressed in the As,Apr,Aal and the inner cells of the cell clusters,while seldom expressed in the somatic cells. CONCLUSIONS: An isolation and culture system of SSCs derived from DBA/2 mice was established. CD44 is highly expressed in the early stage of spermatogonial cell development.

IMB5036 overcomes resistance to multiple chemotherapeutic drugs in human cancer cells through pyroptosis by targeting the KH-type splicing regulatory protein.

AIMS: IMB5036 is a pyridazinone compound with antiproliferative and antitumour activity against hepatoma and pancreatic cancer. In this study, we attempted to identify the target protein of IMB5036 and test its potential for overcoming multidrug resistance and inducing pyroptosis. MATERIALS AND METHODS: We examined the effects of IMB5036 on cancer cells by in vitro assays, a molecular docking model and in vivo tumour models. We performed pull-down experiments using biotinylated IMB5036 and identified the binding proteins. Gene knockdown were used to investigate the oncogenic role of KH-type splicing regulatory protein (KSRP). Western blot was used to detect for mechanism-associated molecules. KEY FINDINGS: IMB5036 could overcome resistance to multiple chemotherapeutic drugs at the cellular level and in vivo. Furthermore, IMB5036 was not a P-glycoprotein (P-gp) substrate and downregulated the expression of P-gp. We identified KSRP as a binding protein of IMB5036. The knockdown of KSRP inhibited the proliferation of MCF7 and MCF7/adriamycin (MCF7/ADR) cells. In addition, IMB5036 induced pyroptosis in both MCF7 and MCF7/ADR cells via KSRP. SIGNIFICANCE: We found IMB5036 binds to KSRP and overcomes multidrug resistance via gasdermin E (GSDME)-dependent pyroptosis.

[Inhibition of tumor cell invasion and induction of apoptosis by ubenimex].

This study is to investigate the effects of ubenimex on tumor cell invasion and apoptosis, dose relationship and mechanism. Immunofluorescence staining was performed to detect the expression of CD13 in HT-1080 cells. MTT assay was used to analyze the effect of ubenimex on cell proliferation. Annexin V-EGFP/PI was used to detect apoptotic cells by flow cytometry. Cell cycle was analyzed using flow cytometry. Ala-pNA was used as substrate to evaluate the effect of ubenimex on the aminopeptidase activity. Transwell assay was used to analyze the effect of ubenimex on cell invasion and migration ability. Western blotting was used to detect the expression level of CD13. MMP activity was analyzed using gelatin zymography. The results showed that ubenimex at high concentration inhibited the proliferation of HT-1080 cells (IC50: 3.8 mg x mL(-1)), and induced cell apoptosis. Cell cycle was blocked at G1 phase. Ubenimex at low concentration inhibited the aminopeptidase activity of HT-1080 cells (IC50: 8.3 microg x mL(-1)) and inhibited cell invasion, but it had no effects on the cell migration and proliferation. Ubenimex had no effects on CD13 expression and MMP activity. In conclusion, ubenimex at low concentration can inhibit the invasion ability of tumor cells by directly inhibiting the aminopeptidase activity; ubenimex at high concentration can inhibit the proliferation of tumor cells and induce cell apoptosis by a CD13-independent pathway.

Mithramycin suppresses tumor growth by regulating CD47 and PD-L1 expression.

Mithramycin A (MIT) has reacquired extensive research attention due to its anti-solid tumor activity and improved pharmacological production. Mechanismly, MIT was broadly used as a c-Myc inhibitor, and c-Myc regulated CD47 and PD-L1 expression which has been demonstrated. However, how MIT affects immune check-point molecules remains unknown. In this study, we found CD47 expression was higher in melanoma of pan-tissue array. MIT inhibited CD47 expression both in mRNA and protein level in melanoma cells (SK-MEL-28 and B16). MIT inhibited c-Myc, Sp-1 and CD47 expression in a concentration-dependent way. MIT inhibited the surface CD47 expression and promoted the phagocytosis of SK-MEL-28 cells by THP-1 cells. We found MIT inhibited tumor growth in melanoma allograft mice and CD47 expression in tumor mass. We also found MIT upregulated PD-L1 expression in cancer cells possibly via inhibiting PD-L1 ubiquitination, increasing ROS and IFN-γ. Combination of MIT and anti-PD-1 antibody showed enhanced antitumor activity compared to MIT and anti-PD-1 antibody alone in MC38 allograft mice. Using immune checkpoint array we found MIT inhibited expression of FasL and Galectin3. These results suggest that MIT inhibits CD47 expression, while improves PD-L1 expression. Furthermore, the combination of MIT and anti-PD-1 antibody exerts potent antitumor effect.

IMB5036 inhibits human pancreatic cancer growth primarily through activating necroptosis.

IMB5036 is a novel pyridazinone compound with potent cytotoxicity. In this study, we reported its antitumour activity against pancreatic cancer and the underlying mechanism. We found that IMB5036 induced rapid cell swelling and increased membrane permeability in pancreatic cancer cells. IMB5036 increased the ratio of PI+ cells, which could be rescued by necroptosis inhibitor. Furthermore, MLKL inhibitor NSA attenuated the killing effect of IMB5036 on pancreatic cancer cells. IMB5036 stimulated translocation of MLKL and p-MLKL from cytoplasm to cell membrane. IMB5036 upregulated the level of p-RIPK1, p-RIPK3, and p-MLKL. At the same time, IMB5036 also partially activated apoptosis and pyroptosis. IMB5036 inhibited tumour growth in pancreatic xenograft. IMB5036 induced larger necrosis area, increased p-MLKL level, and inhibited Ki67 expression in tumour mass. The study indicates that IMB5036 inhibits human pancreatic cancer growth primarily activating necroptosis.

The Effects of the Application of the "One Disease, One Product" Program to Nursing Practice for Patients After the Resection of Pituitary Adenomas Using the Endonasal Transsphenoi.

OBJECTIVE: To explore the application and effect of the "one disease, one product" project to the nursing care of patients who have undergone pituitary tumour surgery using the nasal sphenoid approach. METHODS: This is a prospective research study. In a standard treatment control study, 132 patients undergoing transnasal pituitary tumour surgery were divided into the control group (n = 71) and the observation group (n = 61). The control group was given routine pituitary tumor care, and the "one disease, one product" nursing model was used on the experimental group. The anxiety level of patients, the incidence of postoperative complications, postoperative hospitalization, and levels of satisfaction and capability of group members were measured between the control and experimental groups. RESULTS: There was no difference in the level of anxiety between the 2 groups before admission (P = 0.634). The anxiety level of the patients in the observation group decreased after the "one disease, one product" nursing intervention (P = 0.012), but in the control group, it did not decrease significantly (P = 0.149), and the anxiety level in the control group was significantly higher than in the observation group on day 1 preoperatively (P < 0.001). CONCLUSIONS: "One disease, one product" nursing can reduce the preoperative anxiety and postoperative satisfaction of pituitary adenoma surgery patients through the sphenoid sinus approach. It is worthy of popularization and application in pituitary adenoma resection through the sphenoid sinus approach.

IMB5476, a novel microtubule inhibitor, induces mitotic catastrophe and overcomes multidrug resistance in tumors.

IMB5046 is a nitrobenzoate microtubule inhibitor we reported previously. During screening of its structural analogues, we identified a novel compound IMB5476 with increased aqueous solubility. Here, its antitumor activity and the underlying mechanism were investigated. IMB5476 disrupted microtubule networks in cells and arrested cell cycle at G2/M phase. It inhibited purified tubulin polymerization in vitro. Competition assay indicated that it bound to tubulin at the colchicine pocket. Further experiments proved that it induced cell death by mitotic catastrophe and apoptosis. Notably, it was a poor substrate of P-glycoprotein and exhibited potent cytotoxicity against drug-resistant tumor cells. In addition, IMB5476 could inhibit angiogenesis in vitro. IMB5476 also inhibited the growth of drug-resistant KBV200 xenografts in mice. Conclusively, our data reveal a novel nitrobenzoate microtubule inhibitor with improved aqueous solubility and can overcome multidrug resistance.

[Comparison between biliary cirrhosis and normal porcine liver treated with microwave ablation using a cooled-tip electrode].

OBJECTIVE: To elucidate the difference in both in vivo and ex vivo microwave ablation in a biliary cirrhotic porcine liver model using a cooled-tip antenna. METHODS: Two months after biliary ductal ligation, liver biopsy was performed to confirm the establishment of biliary cirrhosis in 4 Tibetan miniature pigs. Microwave ablation with cooled-tip antenna was conducted under laparotomy using 70 W for five minutes in the experimental group (4 pigs). The control group (2 pigs) also received microwave ablation using the same settings but no surgery. Both in-vivo and ex-vivo ablations were performed in the two groups. Morphological and pathological characteristics of the ablation areas were compared. Paired comparison among the groups were conducted using t-test. RESULTS: In the cirrhotic liver group, after ablation at 70 W for five minutes, the short and long axes and volume of in vivo ablation areas were (1.90 ± 0.10) cm, (2.95 ± 0.12) cm, and (6.0 ± 0.8) cm(3) compared to (2.08 ± 0.08) cm, (3.08 ± 0.75) cm, and (7.0 ± 0.5) cm(3) of ex vivo ablation. In the normal liver group the dates were (2.04 ± 0.05) cm, (3.14 ± 0.11) cm and (6.8 ± 0.5) cm(3); (2.30 ± 0.18) cm, (3.60 ± 0.08) cm and (10.0 ± 1.7) cm(3), respectively. In vivo ablation area was smaller than ex vivo ablation area in terms of short and long axes and volume (P = 0.028 0.026, 0.008, respectively). With the same ablation settings, both in vivo and ex vivo ablation areas in normal pig liver were larger than their counterparts in cirrhotic liver in terms of the short and long axes and volume (P = 0.019, P = 0.000; P = 0.024, P = 0.036, respectively), but the differences in the short axes of in vivo and ex vivo ablation areas failed to reach significance. CONCLUSION: Both in vivo and ex vivo ablation areas in biliary cirrhotic pig liver were smaller than their counterparts in normal pig liver suggesting that, the ablation time or power should be relatively prolonged to enlarge the ablation zone within cirrhotic liver in order to prevent incomplete ablation with viable residual tumor.

PCA3 gene expression in prostate cancer tissue in a Chinese population: quantification by real-time FQ-RT-PCR based on exon 3 of PCA3.

Prostate cancer (PCa) is the second most common cancer in men, and its incidence is still increasing. PCA3 is the most prostate cancer specific biomarker. Here we confirmed that both exon 3 and exon 4 are in the prostate-specific region of the PCA3 gene, and established the methodology of real-time fluorescent quantitative RT-PCR (FQ-RT-PCR) detecting PCA3 mRNA with primer spanning exons 1 and 3, and evaluated its clinical utility in a Chinese population. What disclosed that PCA3 mRNA is prostate cancer specific and shows increased expression in prostate cancer. It could be a reliable molecular marker in prostate cancer diagnosis. Exon 3-based real-time FQ-RT-PCR may prove useful in prostate cancer diagnosis, given that the associated primer would span only exons 1 and 3, relative to other models spanning exons 1 to 4. A shorter amplicon would not only enhance the efficiency of real-time FQ-RT-PCR, but may also simplify the quantification of PCA3 mRNA.

Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression.

A novel protein that associates with interphase nucleus and mitotic apparatus (INMAP) was identified by screening HeLa cDNA expression library with an autoimmune serum followed by tandem mass spectrometry. Its complete cDNA sequence of 1.818 kb encodes 343 amino acids with predicted molecular mass of 38.2 kDa and numerous phosphorylation sites. The sequence is identical with nucleotides 1-1800 bp of an unnamed gene (GenBank accession no. 7022388) and highly homologous with the 3'-terminal sequence of POLR3B. A monoclonal antibody against INMAP reacted with similar proteins in S. cerevisiae, Mel and HeLa cells, suggesting that it is a conserved protein. Confocal microscopy using either GFP-INMAP fusion protein or labeling with the monoclonal antibody revealed that the protein localizes as distinct dots in the interphase nucleus, but during mitosis associates closely with the spindle. Double immunolabeling using specific antibodies showed that the INMAP co-localizes with alpha-tubulin, gamma-tubulin, and NuMA. INMAP also co-immunoprecipitated with these proteins in their native state. Stable overexpression of INMAP in HeLa cell lines leads to defects in the spindle, mitotic arrest, formation of polycentrosomal and multinuclear cells, inhibition of growth, and apoptosis. We propose that INMAP is a novel protein that plays essential role in spindle formation and cell-cycle progression.

DBDx-based drug combinations show highly potent therapeutic efficacy against human pancreatic cancer xenografts in athymic mice.

Previous studies have shown that DBDx, a combination consisting of dipyridamole, bestatin and dexamethasone is highly effective against several cancer xenografts in athymic mice. Here the therapeutic effects of DBDx and its combination with gemcitabine or capcitabine against human pancreatic cancer xenografts and the mechanism were studied. In vivo experiments performed in athymic mice showed that the antitumor efficacy of DBDx was much stronger than that of gemcitabine or capecitabine alone. Notably, the combination of DBDx and gemcitabine or capcitabine further enhanced the efficacy. In the case of DBDx (242 mg/kg) plus gemcitabine (100 mg/kg), tumor weight decreased about 97.7%, and tumor sizes were shrinking during the treatment. In the case of DBDx (242 mg/kg) plus capecitabine (718.7 mg/kg), tumor weight decreased about 94.9%. Moreover, DBDx and its combinations obviously prolonged theoverall survival of mice compared with gemcitabine or capcitabine alone. DBDx-based drug combination therapy showed no obvious systematic toxicity. The gene expression profile analysis showed that the genes changed by DBDx were related to immune system and tumor vasculature. The result of protein array showed that the changed proteins in the serum of treated mice were related to immune and inflammation system. These results show that DBDx-based drug combinations, a new strategy which integrates the use of low-cytotoxic drugs and cytotoxic chemotherapeutics, are highly effective regimens against human pancreatic cancer in athymic mice at well tolerated doses. DBDx-based drug combination therapy might provide new options for the treatment of pancreatic cancer.

Dexamethasone enhances the antitumor efficacy of Gemcitabine by glucocorticoid receptor signaling.

Gemcitabine (Gem) is currently used as the first-line therapy for liver and pancreatic cancer but has limited efficacy in most cases. Dexamethasone (Dex) have been applied as a chemoprotectant and chemosensitizer in cancer chemotherapy. This study further explored the potential of combination of Gem and Dex and tested the hypothesis that glucocorticoid receptor signaling is essential for the synergistic antitumor activity. In the HepG2 and AsPC-1 xenograft models, the combination treatment showed a significantly synergistic antitumor activity. Immunohistochemistry of post-treatment tumors showed a significant decrease in proliferation and angiogenesis as compared to either of the treatments alone. Dex alone and the combination with Gem inhibited the expression of glucocorticoid receptor. The combination of Dex and Gem showed synergistic cytotoxicity in cell lines in vitro. The antiproliferative synergism is prevented by used glucocorticoid receptor (GR) small interfering RNA, demonstrating that the glucocorticoid receptor is required for the antiproliferative synergism of Gem and Dex. The inhibition of glucocorticoid receptor signaling pathway and induction of apoptosis via activation of caspases 3, 8 and 9, PARP, contributed to the synergistic effect of this combination therapy. These results demonstrate that Dex could potentiate the antitumor efficacy of Gem. The synergistic antitumor activity of the combination of Dex and Gem was through glucocorticoid receptor signaling. Taken together, a combination of Dex and Gem shows a significant synergistic antitumor activity and lesser toxicity both in vitro and in vivo and could be a combination chemotherapy for the treatment of highly expression of glucocorticoid receptor patients.