Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited liver cancer.

Background: Oncolytic adenovirus-mediated gene therapy is an emerging strategy for cancer treatment. However, oncolytic adenoviruses are mainly administered locally at tumor site. Intravenous administration of oncolytic adenovirus for cancer gene therapy is a problem that needs to be solved urgently. Methods: We constructed recombinant oncolytic adenovirus KGHV500 carrying anti-p21Ras scFv and employed CIK cells to deliver KGHV500. TUNEL, wound healing, MTT, and Transwell invasion assays were used to determine the anti-tumor efficacy of KGHV500 on liver cancer cells in vitro. Nude mouse xenograft model was used to examine the anti-tumor efficacy of CIK cells combined with KGHV500 in vivo. Furthermore, KGHV500 accumulation in different organs was detected to assess the safety. Results: KGHV500 inhibited the migration, proliferation, invasion, and induced the apoptosis of liver cancer cells. CIK cells carrying KGHV500 reached tumor site and exerted much better anti-tumor efficacy than CIK cells or KGHV500 alone in nude mouse xenograft model. Moreover, we detected KGHV500 and anti-p21Ras scFv in different organs of nude mice, with little effects on the organs. Conclusions: We develop a novel strategy for the treatment of Ras-driven liver cancer by combining CIK cells with oncolytic adenovirus expressing anti-p21Ras scFv. Intravenous injection of CIK cells carrying KGHV500 in vivo significantly inhibits tumor growth, has little effect on normal organs, and is relatively safe.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Inhibition of glioma by adenovirus KGHV500 encoding anti-p21Ras scFv and carried by cytokine-induced killer cells.

Ras gene mutation or overexpression can lead to tumorigenesis in multiple kinds of cancer, including glioma. However, no drugs targeting Ras or its expression products have been approved for clinical application thus far. Adenoviral gene therapy is a promising method for the treatment of glioma. In this study, the human glioma cell line U251 was co-cultured with recombinant adenovirus KGHV500, and the anti-tumor effects of KGHV500 were determined by MTT, scratch test, Transwell invasion, and apoptosis assays. Then, KGHV500 was delivered via the intravenous injection of CIK cells into glioma xenografts. Tumor volume, ki67 proliferation index, apoptosis levels, and anti-p21Ras scFv expression were tested to evaluate targeting ability, anti-tumor efficacy, and safety. We found that the KGHV500 exhibited anti-tumor activity in U251 cells and increased the intracellular expression of anti-p21Ras scFv compared with that in the control groups. CIK cells delivered KGHV500 to U251 glioma cell xenografts and enhanced anti-tumor activity against glioma xenografts compared to that produced by the control treatment. In conclusion, targeting Ras is a useful therapeutic strategy for gliomas and other Ras-driven cancers, and the delivery of anti-p21Ras scFv by recombinant adenovirus and CIK cells may play an essential role in the therapy of Ras-driven cancers.

Comprehensive treatment of rare multiple endocrine neoplasia type 1: A case report.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary disorder caused by mutations of the MEN1 gene. It is characterized by hyperparathyroidism and involves the pancreas, anterior pituitary, duodenum, and adrenal gland. Here, we report a 40-year-old male patient with MEN1 who first manifested as thymic carcinoid, then primary hyperparathyroidism and prolactinoma, and a decade later pancreatic neuroendocrine tumor. CASE SUMMARY: The patient underwent a thymectomy because of the thymic carcinoid 10 years prior and a prolactinoma resection 2 years prior. His sister suffered from prolactinoma. His parents displayed a typical triad of amenorrhea, galactorrhea, and infertility. Computed tomography revealed a strong signal in the upper portion of the left lobes and posterior portion of the right lobes of the thyroid and irregular soft tissue densities of the pancreatic body. Positron emission tomography/computed tomography imaging further showed strong 18F-flurodeoxyglucose uptake in the tail of the pancreatic body and segment IV of the liver. The patient underwent pancreatic body tail resection, pancreatic head mass enucleation, and ultrasound-guided radio-frequency ablation for liver cancer. Pathology results reported neuroendocrine tumor grade 2. Whole exome sequencing revealed a verified pathogenic mutation c.378G>A (p.Trp126*) in the MEN1 gene. The diagnosis of MEN1 was confirmed. At the 1.5-year follow-up, the patient appeared healthy without any sign of reoccurrence. CONCLUSION: The present case may add some insight into the diagnosis and treatment of patients with MEN1.

MicroRNA-527 inhibits TGF-ß/SMAD induced epithelial-mesenchymal transition via downregulating SULF2 expression in non-small-cell lung cancer.

OBJECTIVE: To explore the potential mechanism which miR-527 targeting the heparan sulfate 6-O-endosulfatase (SULF2) regulates TGF-ß/SMAD signaling pathway induced epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer (NSCLC). METHODS: 38 pairs of lung tumor biopsies and corresponding paracancerous biopsies were obtained from NSCLC patients with surgical resection, normal human bronchial epithelial BEAS-2B cells and five NSCLS cell lines were applied for our study. miR-527 and SULF2 expression were determined by qRT-PCR and immunohistochemistry. MiR-527 and SULF2 biological link were predicted by Targetscan.org and tested by dual luciferase. Cells proliferation and apoptosis were respectively detected by EDU staining and flow cytometry. Cells migration was examined by transwell and scratch-wound assay. Expression of proteins related to EMT and TGF-ß/SMAD signaling pathway, such as E-cadherin, N-cadherin, p-Samd3 and p-Smad2, was detected by western blot. RESULTS: miR-527 expression was decreased in lung tumor tissues and NSCLS cell lines, conversely, SULF2 expression was significantly increased. In addition, we found that miR-527 targeted 3'-untranslated regions (3'-UTR) of SULF2 and mediated its expression. Overexpression of miR-527 evidently suppressed NSCLC proliferation, invasion and EMT via TGF-ß/SMAD signaling pathway. Moreover, the silence of SULF2 exhibited a similar effect. CONCLUSION: miR-527 targeting SULF2 down-regulated SULF2 expression, concurrently, suppressed NSCLC epithelial-mesenchymal transition and invasion via inhibiting TGF-ß/SMAD signaling pathway.

CIK cell-based delivery of recombinant adenovirus KGHV500 carrying the anti-p21Ras scFv gene enhances the anti-tumor effect and safety in lung cancer.

PURPOSE: Adenovirus (Ads) is one of the most popular vectors used in gene therapy for the treatment of cancer. However, systemic therapy is limited by circulating antiviral antibodies and poor viral delivery in vivo. In this study, we used cytokine-induced killer (CIK) cells as delivery vehicles of Ads KGHV500 carrying the anti-p21Ras scFv gene to treat Ras gene-related lung cancer and investigate the anti-tumor effect in vitro and in vivo. METHODS: The human lung cancer cell line A549 was employed to investigate the anti-tumor activity of recombinant Ads KGHV500 harboring the anti-p21Ras scFv gene using MTT, wound healing, transwell invasion, and apoptosis assays in vitro. Next, CIK cells were used as delivery vehicles to deliver KGHV500 carrying the anti-p21Ras scFv gene to treat A549-transplanted tumors in nude mice, and viral replication, p21Ras scFv expression, and the therapeutic efficacy were assessed. RESULTS: In vitro studies showed that KGHV500 had potent anti-tumor activity. In addition, in vivo, this combination therapy significantly inhibited the growth of lung cancer xenografts compared with mice treated with KGHV500 alone. KGHV500 and anti-p21Ras scFv were observed in tumor tissue, but were nearly undetectable in normal tissues. CONCLUSIONS: The co-delivery of anti-p21Ras scFv by CIK cells and KGHV500 could increase the anti-tumor effect and safety, and possess considerable advantages for the treatment of Ras-related cancer.

Anti-colorectal cancer effects of anti-p21Ras scFv delivered by the recombinant adenovirus KGHV500 and cytokine-induced killer cells.

BACKGROUND: Colorectal cancer (CRC) is the most common type of gastrointestinal cancer. CRC gene therapy mediated by adenovirus holds great promise for the treatment of malignancies. However, intravenous delivery of adenovirus exhibits limited anti-tumor activity in vivo when used alone. METHODS: In this study, the antitumor activity of the recombinant adenovirus KGHV500 was assessed with the MTT, TUNEL, Matrigel invasion and cell migration assays. To enhance the intravenous delivery of KGHV500 in vivo, cytokine-induced killer (CIK) cells were used as a second vector to carry KGHV500. We explored whether CIK cells could carry the recombinant adenovirus KGHV500 containing the anti-p21Ras single chain fragment variable antibody (scFv) gene into tumors and enhance antitumor potency. RESULTS: Our results showed that KGHV500 exhibited significant antitumor activity in vitro. In the nude mouse SW480 tumor xenograft model, the combination of CIK cells with KGHV500 could induce higher antitumor activity against colorectal cancer in vivo than that induced by either CIK or KGHV500 alone. After seven days of treatment, adenovirus and scFv were detected in tumor tissue but were not detected in normal tissues by immunohistochemistry. Therefore, KGHV500 replicates in tumors and successfully expresses anti-p21Ras scFv in a colorectal cancer xenograft model. CONCLUSIONS: Our study provides a novel strategy for the treatment of colorectal cancer by combining CIK cells with the recombinant adenovirus KGHV500 which carried anti-p21 Ras scFv.

Overexpression of wild-type p21Ras plays a prominent role in colorectal cancer.

Colorectal cancer (CRC) is the most common gastrointestinal type of cancer. The overexpression of Ras proteins, particularly p21Ras, are involved in the development of CRC. However, the subtypes of the p21Ras proteins that are overexpressed and the mutation status remain unknown restricting the development of therapeutic antibodies targeting p21Ras proteins. The present study aimed to investigate the mutation status of ras genes associated with Ras proteins that are overexpressed in CRC and explore whether or not wild-type p21Ras could be a target for CRC therapy. p21Ras expression was examined immunohistochemically in normal colorectal epithelium, benign lesions and malignant colorectal tumor tissues by monoclonal antibody (Mab) KGH-R1 which is able to react with three types of p21Ras proteins: H-p21Ras, N-p21Ras and K-p21Ras. Then, the expression levels of p21Ras subtypes were determined in CRC by a specific Mab for each p21Ras subtype. Mutation status of ras genes in p21Ras-overexpressing CRC was detected by DNA sequencing. There was rare p21Ras expression in normal colorectal epithelium but a high level of p21Ras expression in CRC, with a significant increase from normal colorectal epithelium to inflammatory polyps, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia and invasive colorectal adenocarcinoma, respectively. Overexpression of K-p21Ras was found in all CRC tissues tested, overexpression of N-p21Ras was found in 85.7% of the CRC tissues, while H-p21Ras expression was not found in any CRC tissue. DNA sequencing showed that there were no K-ras mutations in 60% of the K-p21Ras-overexpressing CRC, while 40% of the CRC tissues harbored K-ras mutations. N-ras mutations were not found in any N-p21Ras-overexpressing CRC. Our findings indicate that overexpression of wild-type p21Ras may play a prominent role in the development of CRC in addition to ras mutations and could be a promising target for CRC therapy.

The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB­231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression.

[Chemical constituents of Scrophularia ningpoensis root].

OBJECTIVE: To investigate the chemical constituents from Scrophularia ningpoensis root. METHODS: The compounds were isolated and purified by silica gel and Sephadex LH-20 column chromatography. The structures of these compounds were elucidated on the basis of spectroscopic analysis. RESULTS: Eleven compounds were isolated and identified as vanillin (1), eugenol (2), isoeugenol methyl ether(3), ferulic acid(4), benzoic acid(5), syringic acid(6) ,rhamnopyranosyl vanilloyl(7), syringic acid-4-O-alpha-L-rhamnopyr-anoside(8), beta-sitosterol(9), adenosine (10) and dibutyl phthalate(11). CONCLUSION: Compound 1-3 and 6-8 are obtained from this plant for the first time.

[Comparative analysis of endogenous hormones between two species of Ammopiptanthus seedlings around germination and under Pb2+ stress].

The changes in three endogenous hormones, phytohormones gibberrelic acid (GA3), indoles-3-acid (IAA) and abscisic acid (ABA), were studied around germination and under tress of different density of Pb2+ between two species of Ammopiptanathus. It was found that (1) around germination, in Xinjiang Ammopiptanthus the rate IAA decreased 77.80%, and the rate of ABA decreased 98.90%; and in Mongolia Ammopiptanthus the rate of IAA decreased 75.80%, the rate of ABA decreased 66.20%, and the GA3 contents in both had no big change. (2) With the increase in Pb2+ concentration (20-1 500 mg x L(-1)), the IAA decreased significantly; and only under the high density (more than 1 000 mg x L(-1)) of Pb2+, the GA3 was affected by it; the ABA did not change regularly. So the three endogenous hormone levels of Mongolia Ammopiptanthus were higher than those of Xinjiang Ammopiptanthus. (3) the distributions of Pb2+ in Ammopiptanthus seedlings are the root > stems > leaves. (4) the stress of high concentration of Pb2+ damages irreversibly the leaf cells of Ammopiptanthus. This study can provide the basis of reference data for further research on the growth characteristics, resilience and the mechanism of specific broad-leaved evergreen shrubs-Ammopiptanthus in desertification area.

[Sample targeting under stereomicroscopy can raise the detection rate of early colorectal cancer].

OBJECTIVE: To establish a set of pathological diagnosis method to raise the detection rate of early colorectal cancer. METHODS: All patients with colorectal tumor underwent ordinary electron enteroscopy in 2005. The lesions larger than 10 mm underwent indigo carmine staining and magnifying electron enteroscopy to observe the pit pattern. Endoscopic mucosal resection (EMR) or endoscopic piecemeal resection (EPMR) was performed on the suspected cases of cancer, such as laterally spreading tumor (LST). The resected specimens were stained with cresyl violet and observed by stereomicroscopy to determine the pit patterns. The parts showing the pit patterns associated with early colorectal cancer were targeted and biopsy specimens collected here to undergo pathohistological examination. Routine pathological examination was conducted on the other parts of the same specimen as control. The results of these specimens were compared with those of the specimens collected by ordinary methods from the patients with colorectal tumor in 2004. RESULTS: In 2005 40 patients with colorectal tumor were suspected as with cancer and underwent EMR or EPMR of which 16 were confirmed to be with early stage colorectal cancer, including severe dysplasia by sampling targeting (40%). And the routine pathohistological examination of the randomly collected parts from these same specimens showed 15 cases of mild or moderate dysplasia and only one case of severe dysplasia, with a detection rate of 2.5%, significantly lower than that of the result of sample targeting under stereomicroscopy (P < 0.01). In 2004, out of the 54 patients suspected to be with colorectal cancer only 4 cases of early cancer, including severe dysplasia were detected with a detection rate of 7.4%, significantly than that of the year 2005 (P < 0.01). CONCLUSION: Sample targeting and localized biopsy under stereomicroscopy raises the detection rate of early colorectal cancer.