A dominant variant in DMXL2 is linked to nonsyndromic hearing loss.

PURPOSE: To explore the genetic etiology of deafness in a dominant family with late-onset, progressive, nonsyndromic hearing loss. METHODS: Genome-wide linkage analysis was performed for 21 family members. Candidate pathogenic variants were identified by whole-exome sequencing of selected family members and confirmed by Sanger sequencing of all family members. Cochlear expression of Dmxl2 was investigated by reverse-transcription polymerase chain reaction (RT-PCR) and immunostaining of the organ of Corti from mice. RESULTS: The causative gene was mapped to a 9.68-Mb candidate region on chromosome 15q21.2 (maximum logarithm of the odds score = 4.03) that contained no previously described deafness genes. Whole-exome sequencing identified heterozygous c.7250G>A (p.Arg2417His) in DMXL2 as the only candidate pathogenic variant segregating the hearing loss. In mouse cochlea, expression of DMXL2 was restricted to the hair cells and the spiral ganglion neurons. CONCLUSION: Our data indicated that the p.Arg2417His variant in DMXL2 is associated with dominant, nonsyndromic hearing loss and suggested an important role of DMXL2 in inner ear function.Genet Med advance online publication 22 September 2016.

IF7-Conjugated Nanoparticles Target Annexin 1 of Tumor Vasculature against P-gp Mediated Multidrug Resistance.

Multidrug resistance is the main cause of clinical chemotherapeutic failure. Antiangiogenic cancer therapy with nanomedicine that allows the targeted delivery of antiangiogenic agents to tumor endothelial cells may contribute to innovative strategies for treating multidrug-resistant cancers. In this study, we developed a new nanodrug delivery system (nano-DDS), with improved antiangiogenic efficacy against multidrug resistant human breast cancer MCF-7/ADR cells. Here, the IF7 ligand was a peptide designed to bind the annexin 1 (Anxa 1), a highly specific marker of the tumor vasculature surface, with high affinity and specificity. IF7-conjugated Anxa 1-targeting nanoparticles containing paclitaxel (IF7-PTX-NP) allowed controlled drug release and displayed favorable prolonged circulation in vivo. IF7-PTX-NP was significantly internalized by human umbilical vein endothelial cells (HUVEC) through the IF7-Anxa 1 interaction, and this facilitated uptake enhanced the expected antiangiogenic activity of inhibiting HUVEC proliferation, migration, and tube formation in a Matrigel plug relative to those of Taxol and PTX-NP. As IF7-PTX-NP targeted the tumor vessels, more nanoparticles accumulated in MCF-7/ADR tumors, and more importantly, induced significant apoptosis of the tumor vascular endothelial cells and necrosis of the tumor tissues. Low dose paclitaxel (1 mg/kg) formulated in IF7-PTX-NP showed significant anticancer efficacy, delaying the growth of MCF-7/ADR tumors. The same efficacy was only obtained with an 8-fold dose of paclitaxel (8 mg/kg) as Taxol plus XR9576, a potent P-gp inhibitor. The anticancer efficacy of IF7-PTX-NP was strongly associated with the improved antiangiogenic effect, evident as a dramatic reduction in the tumor microvessel density and pronounced increase in apoptotic tumor cells, with no obvious toxicity to the mice. This nano-DDS, which targets the tumor neovasculature, offers a promising strategy for the treatment of multidrug-resistant cancer.

Luteolin-Loaded Nanoparticles for the Treatment of Melanoma.

Background and Purpose: Luteolin (LUT), a flavonoid found in various plants, has been reported to have potential therapeutic effects in melanoma. However, poor water solubility and low bioactivity have severely restricted the clinical application of LUT. Based on the high reactive oxygen species (ROS) levels in melanoma cells, we developed nanoparticles encapsulating LUT with the ROS-responsive material poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG) to enhance the water solubility of LUT, accelerate the release of LUT in melanoma cells, and further enhance its anti-melanoma effect, providing a viable solution for the application of LUT nano-delivery systems in melanoma therapy. Methods: In this study, LUT-loaded nanoparticles were prepared with PPS-PEG and named as LUT-PPS-NPs. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were applied to determine the size and morphology of LUT-PPS-NPs. In vitro studies were carried out to determine the uptake and mechanism of LUT-PPS-NPs by SK-MEL-28 melanoma cells. According to the CCK-8 assay, the cytotoxic effects of LUT-PPS-NPs on human skin fibroblasts (HSF) and SK-MEL-28 cells were assessed. Apoptosis assays, cell migration and invasion assays, and proliferation inhibition assays with low and normal density plating were also applied to test the in vitro anti-melanoma effect. Additionally, melanoma models were established utilizing BALB/c nude mice and initially evaluated the growth inhibitory impact following intratumoral injection of LUT-PPS-NPs. Results: The size of LUT-PPS-NPs was 169.77 ± 7.33 nm with high drug loading (15.05 ± 0.07%). In vitro, cellular assays confirmed that LUT-PPS-NPs were efficiently internalized by SK-MEL-28 cells and showed low cytotoxicity against HSF. Moreover, LUT released from LUT-PPS-NPs significantly inhibited tumor cell proliferation, migration and invasion. Animal experiments showed that LUT-PPS-NPs inhibited tumor growth more than 2-fold compared with the LUT group. Conclusion: In conclusion, the LUT-PPS-NPs developed in our study enhanced the anti-melanoma effect of LUT.

Antiangiogenic polyketides from Peperomia dindygulensis Miq.

Two new polyketides: 2Z-(heptadec-12-enyl)-4-hydroxy-3,4,7,8-tetrahydro-2H-chromen-5(6H)-one (1) and 2-(heptadec-12-enyl)-5-hydroxy-5,6,7,8-tetrahydrochromen- 4-one (2), together with eleven known compounds: 4-hydroxy-2-[(3,4-methylenedioxy- phenyl)tridecanoyl] cyclohexane-1,3-dione (3), oleiferinone (4), 4-hydroxy-2-[(3,4- methylenedioxyphenyl)undecanoyl]cyclohexane-1,3-dione (5), 4-hydroxy-2-[(11-phenyl- undecanoyl)cyclohexane-1,3-dione (6), proctorione C (7), surinone C (8), 5-hydroxy- 7,8,4'-trimethoxyflavone (9), 5-hydroxy-7,8,3',4'-tetramethoxyflavone (10), 5-hydroxy- 7,3',4'-trimethoxyflavone (11), 5,8-dihydroxy-7,3',4'-trimethoxyflavone (12) and cepharanone B (13) were isolated from the whole plant of Peperomia dindygulensis Miq. Their structures were elucidated by spectroscopic methods, including 2D-NMR techniques. Compounds 2, 3, 5 and 8 inhibited human umbilical vein endothelial cell (HUVEC) proliferation and compounds 5 and 8 sharply suppressed HUVEC tube formation.

Secolignans with antiangiogenic activities from Peperomia dindygulensis.

Two new secolignans, peperomins G and H (1 and 2, resp.), were isolated from the whole plant of Peperomia dindygulensis, together with five known secolignans, peperomin A (3), peperomin E (4), peperomin B (5), 2,3-trans-2-methyl-3-{(3-hydroxy-4,5-dimethoxyphenyl)[5-methoxy-3,4-(methylenedioxy)phenyl]methyl}butyrolactone (6), 2,3-cis-2-(hydroxymethyl)-3-{bis[5-methoxy-3,4-(methylenedioxy)phenyl]methyl}butyrolactone (7). Their structures and configurations were elucidated by spectroscopic methods including 2D-NMR techniques. Antiangiogenic effects of all compounds were evaluated using human umbilical vein endothelial cells (HUVEC) proliferation and tube-formation tests, with compounds 4 and 5 being active in the bioassay. Compounds 4 and 5 induced obvious cell toxicity to HUVEC with IC(50) values of 1.64±0.19 and 8.44±0.4 µM, respectively. Compounds 4 and 5 also exhibited significant HUVEC tube formation-inhibiting activity with IC(50) values of 3.13±0.09 and 6.24±0.12 µM, respectively.

Protection of PC12 cells against superoxide-induced damage by isoflavonoids from Astragalus mongholicus.

OBJECTIVE: To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/ xanthine oxidase (XO)-induced injury to PC12 cells. METHODS: PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isoflavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. RESULTS: Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-beta-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/ XO-induced injury to PC12 cells. Their EC50 values of formononetin and calycosin were 0.05 microg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 microg/mL and 50 microg/mL. Formononetin and calycosin had no significant influence on Bcl-2 or Bax protein contents. CONCLUSION: Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.

Genetic analysis of a Chinese family with members affected with Usher syndrome type II and Waardenburg syndrome type IV.

AIMS: The purpose of this study was to identify the genetic causes of a family presenting with multiple symptoms overlapping Usher syndrome type II (USH2) and Waardenburg syndrome type IV (WS4). METHODS: Targeted next-generation sequencing including the exon and flanking intron sequences of 79 deafness genes was performed on the proband. Co-segregation of the disease phenotype and the detected variants were confirmed in all family members by PCR amplification and Sanger sequencing. RESULTS: The affected members of this family had two different recessive disorders, USH2 and WS4. By targeted next-generation sequencing, we identified that USH2 was caused by a novel missense mutation, p.V4907D in GPR98; whereas WS4 due to p.V185M in EDNRB. This is the first report of homozygous p.V185M mutation in EDNRB in patient with WS4. CONCLUSION: This study reported a Chinese family with multiple independent and overlapping phenotypes. In condition, molecular level analysis was efficient to identify the causative variant p.V4907D in GPR98 and p.V185M in EDNRB, also was helpful to confirm the clinical diagnosis of USH2 and WS4.

Studies of chemical constituents and their antioxidant activities from Astragalus mongholicus Bunge.

OBJECTIVE: To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine (XA)/xanthine oxidase (XO)-induced toxicity in PC12 cells. METHODS: The compounds of Astragalus mongholicus Bunge were isolated by chromatography and the structures were elucidated on the basis of spectral data interpretation. Their antioxidant activities were detected by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in a cell-free system. Meanwhile, the effects against XA/XO-induced toxicity were assessed using MTT assay in PC12 cells. RESULTS: Ten principal constituents were isolated and identified as formononetin (I), ononin (II), calycosin (III), calycosin-7-O-beta-D-glucoside (IV), 9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (V), adenosine (VI), pinitol (VII), daucosterol (VIII), beta-sitoster (IX) and saccharose (X) from Astragalus mongholicus Bunge. The compounds I, III, and IV scavenged DPPH free radicals in vitro. Formononetin and calycosin were found to inhibit XA/XO-induced cell injury significantly, with an estimated EC50 of 50 ng/mL. CONCLUSION: Compound II, VI, and VII are first reported in this plant. Calycosin exhibits the most potent antioxidant activity both in the cell-free system and in the cell system.

Genetic detection of Chinese hereditary nonpolyposis colorectal cancer.

AIM: To explore the germline mutations of the two main DNA mismatch repair genes (hMSH2 and hMLH1) between patients with hereditary non-polyposis colorectal cancer (HNPCC) and suspected (atypical) HNPCC. METHODS: Genomic DNA was extracted from the peripheral blood of the index patient of each family, and germline mutations of hMSH2 and hMLH1 genes were detected by PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing techniques. RESULTS: For PCR-SSCP analysis, 67% (4/6) abnormal exons mobility in typical group and 33% (2/6) abnormal exons mobility in atypical group were recognized. In direct DNA sequencing, 50% (3/6) mutation of MMR genes in typical group and 33% (2/6) mutation of MMR genes in atypical group were found, and 4/6 (66.67%) and 1/6 (16.67%) mutations of hMSH2 and hMLH1 were identified in typical HNPCC and atypical HNPCC, respectively. CONCLUSION: Mutation detection of the patients is of benefit to the analysis of HNPCC and, PCR-SSCP is an effective strategy to detect the mutations of HNPCC equivalent to direct DNA sequence. It seems that there exist more complicated genetic alterations in Chinese HNPCC patients than in Western countries.

[The role of the immunohistochemistry for hMLH1 and hMSH2 with detection of microsatellite instability to identify the kindreds with hereditary nonpolyposis colorectal cancer].

OBJECTIVE: To investigate the specificity and sensitivity of the immunohistochemistry for hMLH1 and hMSH2 with detection of microsatellite instability (MSI) to identify the kindreds with hereditary nonpolyposis colorectal cancer and to analyse its value in clinical practice. METHOD: Specimens of 16 cases with HNPCC and 16 cases with sporadic colorectal cancer were detected by immunostaining with hMLH1 and hMSH2 and MSI was also detected. RESULTS: The specificity and sensitivity of the immunohistochemistry for hMLH1 and hMSH2 were 91.7% and 87.5% respectively. The specificity and sensitivity of MSI were 100% and 75.0%. By combining two methods, the specificity and sensitivity were 91.7% and 93.8% respectively. CONCLUSIONS: By combination of the immunohistochemistry for hMLH1 and hMSH2 and detection of MSI to identify the kindreds with HNPCC, the specificity and sensitivity are improved which is better than to use either of them alone. And it is very easy and cheap that it can be used in clinics.

[Clinicopathological features of typical and non-typical hereditary non-polyposis colorectal cancer and their germline mutation of hMLH1 and hMSH2].

OBJECTIVE: To study the clinicopathological features of the Chinese hereditary non-polyposis colorectal cancer and its germline mutation of hMLH(1) and hMSH(2). METHODS: Thirteen typical Chinese hereditary non-polyposis colorectal carcinoma (HNPC)C kindreds and 19 non-typical HNPCC families were registered and followed up. The germline mutation of the hMLH(1) and hMSH(2) of 12 index cases of 6 typical and 6 non-typical HNPCC were screened by PCR-SSCP. Samples with abnormal mobility were sequenced directly. RESULTS: The average age of typical HNPCC was 47, no difference existed between sexes. Location of the tumors of typical HNPCC represented 44.7% on the right half colon and non-typical HNPCC 65.8% on the rectum. The rate of the metachronos cancer was 11.5%. The 3-, 5-and 10-year survival rate was 64.0%, 45.3% and 31.2% respectively. Among 12 cases, 8 showed abnormal mobility. Except for an intron polymorphism, six exons abnormalities were found in 5 of 12 proband. Sequencing showed 4 missense, 7 insertion and a nonsense mutations. CONCLUSIONS: Chinese HNPCC is early onset, more common on proximal colon and better prognosis. Mutation of hMSH(2) is dominant in the Chinese typical HNPCC, but mutation of hMLH(1) is more common in the non-typical group.

Suppression of colorectal cancer subcutaneous xenograft and experimental lung metastasis using nanoparticle-mediated drug delivery to tumor neovasculature.

Antiangiogenic therapy is a validated approach for colorectal cancer (CRC) treatment. However, diverse adverse effects inevitably appear due to the off-target effect of the approved antiangiogenic inhibitors on the physiological functions and homeostasis. This study was to investigate a new tumor vessel targeting nanoparticulate drug delivery system, F56 peptide conjugated nanoparticles loading vincristine (F56-VCR-NP), for the effective treatment of CRC subcutaneous xenograft and experimental lung metastasis model. The controlled release behavior and in vivo pharmacokinetic profile of F56-VCR-NP were characterized. The tumor vessel targeting and antiangiogenic activity of F56-VCR-NP was evaluated in human umbilical vein endothelial cells (HUVEC, a classical cell model mimicking tumor vascular EC), subcutaneous human HCT-15 xenograft in immunodeficient nude mice, and experimental CT-26 lung metastasis model in immunocompetent mice. The therapeutic efficacy (animal survival and toxicity) was further investigated in the model of CT-26 lung metastasis in mice. F56-VCR-NP could achieve 30-day controlled drug release in PBS (pH 7.4) and exhibited favorable long-circulating feature in vivo. F56-VCR-NP could accurately target the CRC neovasculature and elicit nanoparticle internalization in the tumor vascular EC, where the antiangiogenic VCR-induced dramatic EC apoptosis and necrosis of CRC tissue. F56-VCR-NP significantly prolonged the mouse survival with no obvious toxicity (weight loss and anepithymia) in the CT-26 lung metastasis mice model, and this pronounced antitumor effect was closely related with the decreased microvessel density in the metastases. The present nanoparticle-based targeted antiangiogenic therapy may provide a new promising approach for the therapy of CRC and lung metastasis, which deserves further translational research.

Determination of Raddeanin A in rat plasma by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study.

A simple, rapid and sensitive LC-MS/MS analysis method was developed and validated for the determination of Raddeanin A (RA) in rat plasma. Protein precipitation with three volumes of methanol as the precipitation reagent was used as the sample preparation method. The analysis process was performed on a Thermo Syncronis C18 column with the mobile phase of methanol-water (containing 5mM ammonium formate, pH 2.2) (85:15, v/v). RA and glycyrrhetinic acid (internal standard) were monitored under negative electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of RA and IS were 2.1 min and 3.5 min, respectively. The limit of detection was 5 ng/mL and the linear range was 50-50,000 ng/mL. The intra-day and inter-day precision was 1.87-2.94% and 3.25-5.36%, and the intra-day and inter-day accuracy ranged from 5.9% to 10.5% and 5.6% to 11.1%, respectively. The absolute recovery was above 90.3%. The method has been successfully translated to the pharmacokinetic study of RA in rats after intravenous and intraperitoneal administration (0.75 mg/kg).

The use of nanoparticulate delivery systems in metronomic chemotherapy.

Metronomic chemotherapy aiming at inhibiting tumor angiogenesis with conventional chemotherapeutics is a promising strategy for antiangiogenic cancer therapy. However, current metronomic chemotherapy mainly focuses on free small-molecule drugs, without any effort to achieve tumor-specific biodistribution, which may lead to long-term toxicity concerns. Metronomic chemotherapy using nanoparticulate drug delivery system (DDS) offers significant upside to reduce off-target side effects, decrease accumulated dose, and enhance the efficacy of tumor vessel targeting without compromising antitumor efficacy; but there has been a lack of thorough experimental data describing the targeted metronomic chemotherapy. Here, we develop a new nanoparticulate DDS, SP5.2 peptide conjugated, Flt-1 (VEGFR-1) targeted nanoparticles for docetaxel (SP5.2-DTX-NP), as a model for the investigation of targeted metronomic chemotherapy with respect to both antitumor efficacy and toxicity. The results demonstrate that metronomic SP5.2-DTX-NP exerts antitumor activity mainly through the antiangiogenic effect of docetaxel, which is specifically delivered into the tumor vascular endothelial cells through the nanoparticle internalization mediated by the interaction of SP5.2 and over-expressed Flt-1 receptors on tumor vessels. Moreover, the antitumor efficacy of targeted metronomic chemotherapy is better than that of the treatment with the DDS given in the maximum tolerated dose (MTD) regimen, which is shown in significantly prolonged mice survival and minimal drug-associated toxicity (bone marrow suppression, hematological toxicity, and mucosal injury of small intestine). The present research reveals and highlights the significance of targeted metronomic therapy with nanoparticulate DDS in antiangiogenic cancer therapy.

Nanoparticle-mediated drug delivery to tumor neovasculature to combat P-gp expressing multidrug resistant cancer.

Anticancer drug resistance is a common intractable obstacle in clinical cancer chemotherapy. Here, we hypothesize that antiangiogenic cancer therapy through the targeted delivery of antiangiogenic agents to the tumor endothelial cells (EC), not the resistant cancer cells, may have the potential of combating multidrug resistant cancer. The K237 peptide-conjugated paclitaxel loaded nanoparticles (K237-PTX-NP), which can target KDR receptors highly expressed in the tumor vasculature, were fabricated for this investigation and the human colorectal adenocarcinoma HCT-15 with naturally expressed P-gp on the cell surface was adopted as the resistant tumor model. The human umbilical vein endothelial cells (HUVEC, a classical cell model mimicking tumor EC) were much more sensitive, in the cytotoxicity and apoptosis test, to K237-PTX-NP than Taxol and non-targeted PTX-NP. The enhanced antiangiogenic feature of K237-PTX-NP can be ascribed to the active internalization mediated by the interaction of K237 and KDR specifically highly expressed on the HUVEC, and the significantly extended intracellular drug retention. The tumor vessel targeting of K237-PTX-NP led to increased nanoparticle accumulation in HCT-15 tumors, and more importantly, induced significant apoptosis of tumor vascular EC and necrosis of tumor tissues. Low dose paclitaxel formulated in K237-PTX-NP (1 mg/kg) achieved significant anticancer efficacy of inhibiting the growth of HCT-15 tumors, but the same efficacy could be only obtained with 8 fold dose paclitaxel (8 mg/kg) in Taxol plus XR9576, a potent P-gp inhibitor. The anticancer efficacy of K237-PTX-NP was well related with the improved antiangiogenic effect shown in the dramatically decreased intratumoral microvessel density and pronouncedly increased apoptotic tumor cells, and such approach did not lead to obvious toxicity in the mice. These results suggest that the nanoparticles targeting drug to tumor neovasculature may be a promising strategy for the treatment of multidrug resistant cancer.

Antiangiogenic triterpenes isolated from Chinese herbal medicine Actinidia chinensis Planch.

Actinidia chinensis Planch. is a famous Chinese herbal medicine to treat many diseases such as cancers. Triterpenes, polyphenols and anthraquinones are normally considered as the main constituents for its effects. In this study, eleven known triterpenes were isolated from the root of Actinidia chinensis., and were examined for its antiangiogenic activities. Their structures were elucidated by comprehensive spectroscopic methods, including IR, UV, HR-ESI-MS, and 1D and 2D NMR techniques. The eleven compounds are following: 2α,3α,19-trihydroxyurs-12-en-28-oic acid (1), 2α,3ß-dihydroxyurs-12-en-28-oic acid (2), 2α,3α,23-trihydroxyurs-12-en-28-oic acid (3), asiatic acid (4), ursolic acid (5), 2α,3ß,19,24-tetrahydroxyurs-12-en-28-oic acid (6), 2α,3ß,19-trihydroxyolean-12-en-28-oic acid (7), 2α,3α,24-trihydroxyolean-12-en-28-oic acid (8), oleanolic acid (9), 3ß-O-acetyloleanolic acid (10), 2α,23-dihydroxylmicromeric acid (11). All these compounds were evaluated with respect to their antiangiogenic activities utilizing the assays of human umbilical vein endothelial cells (HUVEC) proliferation and tube formation and Ursolic acid (used as control) and compounds 2, 3, 4, and 8 exhibited significant, dose-dependently, antiangiogenic activity in the tested concentration range. Our findings suggest that antitumor action of Actinidia chinensis Planch. is partly via inhibiting tumor angiogenesis by triterpenes, and compounds 2, 3, 4, and 8 as the novel potential antiangiogenic agents are worthy of further translational research.

Dichloroacetate shifts the metabolism from glycolysis to glucose oxidation and exhibits synergistic growth inhibition with cisplatin in HeLa cells.

The unique bioenergetic feature of cancer, aerobic glycolysis or the Warburg effect, is an attractive therapeutic target for cancer therapy. Reversing the glycolytic phenotype may trigger apoptosis in tumor cells. Recently, dichloroacetate (DCA) was proven to produce significant cytotoxic effects in certain tumor cells through this distinct mechanism. In this study, the effect of DCA on the metabolism of cervical cancer HeLa cells was explored and its synergistic growth inhibition with cisplatin was also evaluated. The intracellular changes in HeLa cells following DCA exposure were analyzed through cell viability, intracellular H2O2 and pH levels, mitochondrial membrane potential (MMP), expression of apoptotic proteins and Kv1.5 channel, and intracellular-free Ca2+ concentration ([Ca2+]i). For the evaluation of combination chemotherapy, HeLa cells were treated with a combination of DCA and cisplatin at various concentrations for 48 h. Cell viability was determined by CCK-8 assay and the synergy of the two agents was evaluated using the R index method. DCA shifted the metabolism of HeLa cells from aerobic glycolysis to glucose oxidation as shown by the increased intracellular H2O2 and pH levels. The change of the metabolism modality led to a drop in MMP and the increase of apoptotic proteins (caspase 3 and 9). The increased Kv1.5 expression and decreased [Ca2+]i established a positive feedback loop that resulted in reduced tonic inhibition of caspases. Combination chemotherapy of DCA and cisplatin exhibited a significant synergy in inhibiting the proliferation of HeLa cells. The specific apoptotic mechanism of DCA as distinguished from the cisplatin may be partly responsible for the synergy and further in vivo study on combination chemotherapy of the two agents in cervical cancer xenografts in mice is warranted.

Peptide-conjugated biodegradable nanoparticles as a carrier to target paclitaxel to tumor neovasculature.

Antiangiogenic cancer therapy can be achieved through the targeted delivery of antiangiogenic agents to the endothelial cells of tumor neovasculature. In the present study, we developed a drug delivery system (DDS), nanoparticles conjugated with K237-(HTMYYHHYQHHL) peptides for tumor neovasculature targeting drug delivery. Paclitaxel, a chemotherapeutic agent with potent antiangiogenic activity, was used as a prototype drug. We synthesized the aldehyde poly(ethylene glycol)-poly(lactide) (aldehyde-PEG-PLA) block copolymer by ring opening polymerization. The nanoparticles loading paclitaxel (PTX-NP) were fabricated using the O/W emulsion and evaporation technique. K237 ligand, a peptide that can bind to the KDR receptors predominantly expressed on the surface of tumor neovasculature endothelial cells with high affinity and specificity and inhibit the VEGF-KDR angiogenic signal pathway, was conjugated to the aldehyde group of PEG chain using the N-terminal PEGylation technique. The K237 conjugated paclitaxel-loaded nanoparticles (K237-PTX-NP) had a hydrodynamic diameter of 150 nm. The K237 density on nanoparticle surface was 474 and the mean distance between two neighboring PEG chains linked to K237 peptide was 12 nm. The K237 conjugated nanoparticles could be significantly internalized by human umbilical vein endothelial cells (HUVEC) through the K237-KDR interaction, and this facilitated uptake led to the expected enhanced antiangiogenic activity shown by HUVEC proliferation, migration and tube formation compared to cells treated with the commercial formulation Taxol and PTX-NP. The long-circulating property and the K237 ligand of K237-PTX-NP warranted rapid, long-term, and accurate in vivo tumor neovasculature targeting, and thereafter the significant apoptosis of tumor neovasculature endothelial cells and necrosis of tumor tissues of MDA-MB-231 breast tumors implanted in female BLAB/c nude mice. This nanoparticulate DDS offers a new strategy for paclitaxel chemotherapy application and it could also be used to carry other chemotherapeutic drugs, genes, and proteins with antiangiogenic activity for antiangiogenic cancer therapy.

[Transsacral resection for presacral tumors].

OBJECTIVE: To explore the operation indication and safety of presacral tumor. METHODS: Clinical data of 36 patients with presacral tumor from November 1990 to May 2006 treated in our hospital, in whom 23 patients underwent trans-sacral operation, were analyzed retrospectively. RESULTS: The operation time was from 43 to 210 min (average 94 min). The volume of blood loss was from 30 to 2000 ml (average 350 ml). Hospital stay was from 8 to 16 days (average 10.7 days). There were 13 different pathology types of tumors in the 36 patients including 26.4% of malignancy. Complications of trans-sacral operation included 1 case of ureteral damage, 1 case of sacral wound hernia, 1 case of presacral abscess who was healed by sigmoid stoma and wound drainage. CONCLUSION: Trans-sacral resection of low presacral tumor is safe and effective with less trauma, less bleeding and quick recovery.

Anti-4-1BB scFv immunogene therapy and low dose cyclophosphamide exhibit a synergistic antitumor effect in established murine lung tumors.

T-cell costimulatory molecules such as 4-1BB may provide a distinct and important signal for promoting positive immune regulation. 4-1BB is thought to have potential use as a cancer immunotherapeutic drug. In our previous study, a nonreplicative adenovirus (Ad.4-1BB scFv) carrying single-chain Fv fragments (scFv) specific for the 4-1BB gene (anti-4-1BB scFv) possessed remarkable in vivo anti-hepatoma efficacy. However, monotherapy achieved by triggering 4-1BB signaling was not sufficient to induce eradicative antitumor activities in low immunogenic tumors. It is of great interest to explore any possible synergistic antitumor effect of 4-1BB signaling combined with low dose cyclophosphamide (CTX), which is well documented to inhibit the suppressive capability of regulatory T-cells in mice and humans. In the present study, recombinant nonreplicative adenoviruses carrying an anti-4-1BB scFv gene were generated, characterized and explored for their stimulation of antilung tumor (TC-1) immunity in immunocompetent C57BL/6 mice. Compared to adenovirus and cyclophosphamide alone, adenovirus-mediated anti-4-1BB scFv in combination with low dose CTX treatment could obviously augment the antitumor activity, in which some established TC-1 tumors were eradicated and the survival of mice was significantly extended. This synergistic antitumor effect could be largely attributed to the depletion of T regulatory cells induced by low dose CTX. These findings may provide a new and promising strategy for immunogene therapy against cancer.