Advancements in ovarian cancer immunodiagnostics and therapeutics via phage display technology.

Ovarian cancer, ranking as the seventh most prevalent malignancy among women globally, faces significant challenges in diagnosis and therapeutic intervention. The difficulties in early detection are amplified by the limitations and inefficacies inherent in current screening methodologies, highlighting a pressing need for more efficacious diagnostic and treatment strategies. Phage display technology emerges as a pivotal innovation in this context, utilizing extensive phage-peptide libraries to identify ligands with specificity for cancer cell markers, thus enabling precision-targeted therapeutic strategies. This technology promises a paradigm shift in ovarian cancer management, concentrating on targeted drug delivery systems to improve treatment accuracy and efficacy while minimizing adverse effects. Through a meticulous review, this paper evaluates the revolutionary potential of phage display in enhancing ovarian cancer therapy, representing a significant advancement in combating this challenging disease. Phage display technology is heralded as an essential instrument for developing effective immunodiagnostic and therapeutic approaches in ovarian cancer, facilitating early detection, precision-targeted medication, and the implementation of customized treatment plans.

Surgical site infection after intracorporeal and extracorporeal anastomosis in laparoscopic left colectomy for colon cancer: a multicenter propensity score-matched cohort study.

BACKGROUND: Intracorporeal anastomosis (IA) is associated with reduced surgical site infection (SSI) and other postoperative complications in laparoscopic right colectomy (LRC). However, evidence is inadequate for IA in laparoscopic left colectomy (LLC). This study aimed to determine the effect of IA and extracorporeal anastomosis (EA) on SSI and other short-term postoperative complications in LLC. METHODS: In this retrospective multicenter propensity score-matched (PSM) cohort study, we enrolled consecutive patients who underwent LLC with IA (TLLC/IA) and laparoscopic-assisted left colectomy with EA (LALC/EA) at two medical centers between January 2015 and September 2021. Propensity score matching with a 1:2 ratio was employed. The primary outcome was SSI occurrence. Secondary outcomes were operating time, intraoperative hemorrhage, other postoperative complications, and pathological outcomes. RESULTS: Overall, 574 and 99 patients received LALC/EA and TLLC/IA, respectively. After PSM, 84 patients with TLLC/IA were matched with 141 patients with LALC/EA. Thirty patients (13.3%) patients experienced SSI (17.0% in LALC/EA vs 7.1% in TLLC/IA). IA was associated with a reduced risk of overall SSI and superficial/deep SSI compared with EA after PSM, with OR of 0.375 (95% CI, 0.147-0.959, P = 0.041). and 0.148 (95% CI, 0.034-0.648, P = 0.011), respectively. Multivariate analysis of unmatched patients indicated similar results. In the analysis of secondary outcomes, LALC/EA may have a shorter operating time (absolute mean difference - 13.41 [95% CI, - 23.76 to - 3.06], P = 0.002) and a higher risk of intraoperative hemorrhage (absolute risk difference 4.96 [95% CI, - 0.09 to 9.89], P = 0.048). CONCLUSIONS: IA in LLC is associated with a reduced risk of overall SSI and superficial/deep SSI. However, it may require a longer operating time.

Surgical site infection after intracorporeal anastomosis for left-sided colon cancer: study protocol for a non-inferiority multicenter randomized controlled trial (STARS).

BACKGROUND: Surgical site infection (SSI), as one of the most common hospital-acquired infection, is usually associated with increased morbidity, mortality, and health care burden. SSI is a significant perioperative complication after colon cancer surgery, particularly for left-sided colon cancer. This paper describes the background and design of the "Surgical Site Infection after intracorporeal anastomosis for Left-sided Colon Cancer: study protocol for a non-inferiority multicenter Randomized Controlled Trial (STARS)." The STARS trial aims to compare the incidence of SSI after intracorporeal anastomosis and extracorporeal anastomosis after radical resection of colon cancer and to explore the risk factors of SSI. METHODS: A total of 354 left colon cancer patients from 8 hospitals in China will be enrolled in this multi-center randomized controlled study. The primary outcome of this study is the incidence of SSI 30 days after left-sided colon cancer surgery. Secondary outcome measures include operation time, blood loss, conversion rate, incidence of perioperative complications, completeness of resection, number of lymph nodes collected and postoperative recovery characteristics, 3-year disease-free survival, and 5-year overall survival. The first patient was enrolled in January 2021. DISCUSSION: To our knowledge, this is the first prospective multicenter study to investigate whether there is a difference in the SSI incidence after intracorporeal and extracorporeal anastomosis for left-sided colon cancer in China. The results may provide more evidence that supports performing total laparoscopic left-sided colon cancer surgery. TRIAL REGISTRATION: The trial has been registered on ClinicalTrials.gov website (ID: NCT04201717). Registered on September 22, 2020.

The Medial Border of Laparoscopic D3 Lymphadenectomy for Right Colon Cancer: Results from an Exploratory Pilot Study.

BACKGROUND: Opinions vary on the medial border of D3 lymphadenectomy for right colon cancer. Most surgeons place the medial border along the left side of the superior mesenteric vein, but some consider the left side of the superior mesenteric artery as the medial border. OBJECTIVES: This study investigated the clinical outcomes of laparoscopic D3 lymphadenectomy for right colon cancer with the medial border along the left side of superior mesenteric artery. DESIGN: This was a retrospective study. SETTINGS: The study was conducted in specialized colorectal cancer department of 5 tertiary hospitals. PATIENTS: Patients receiving laparoscopic D3 lymphadenectomy for right colon cancer from January 2013 to December 2018 were included. MAIN OUTCOME MEASURES: After propensity score matching, 307 patients receiving laparoscopic D3 lymphadenectomy along the left side of the superior mesenteric artery were assigned to the superior mesenteric artery group and 614 patients were assigned to the superior mesenteric vein group. Univariate, multivariate, and Kaplan-Meier analyses were performed to assess the clinical data. RESULTS: The short-term outcomes were similar between the 2 groups; however, the superior mesenteric artery group had a higher rate of chylous leakage (p < 0.001). More lymph nodes were harvested from the superior mesenteric artery group than from the superior mesenteric vein group (p = 0.001). The number (p = 0.005) of metastatic lymph nodes and the lymph node ratio (p = 0.041) in main nodes were both higher in the superior mesenteric artery group. The 2 groups had similar long-term survival, but the superior mesenteric artery group tended to show better disease-free survival in patients with stage disease III (p = 0.056). LIMITATIONS: This was a retrospective, nonrandomized study. CONCLUSION: Laparoscopic D3 lymphadenectomy along the left side of the superior mesenteric artery, except for a higher rate of chylous leakage, had short-term outcomes comparable to the superior mesenteric vein group. The superior mesenteric artery group tended to achieve better disease-free survival in patients with stage III disease, but further study is required to better elucidate differences in these approaches because risks/benefits do exist.

Knowledge, attitudes, practices, and related factors of low anterior resection syndrome management among colorectal surgery nurses: a multicenter cross-sectional study.

PURPOSE: With the widespread development of low and ultra-low rectal sphincter-preservation surgery, low anterior resection syndrome, a new clinical challenge, has received increased attention. As the principal practitioners of this syndrome management, colorectal surgery nurses require sufficient relevant knowledge and skills, but few studies have been conducted of their actual ability and practice of managing the syndrome. The study adopted a knowledge, attitude, and practice model to evaluate low anterior resection syndrome management among colorectal surgery nurses. METHOD: A multicenter cross-sectional study was conducted, in which 361 registered nurses in colorectal surgery from 6 hospitals in 4 cities were enrolled. A structured paper questionnaire was used to collect demographics, scale scores for knowledge, attitudes and management practice, and training needs. RESULTS: Participants scored poorly in knowledge, attitude, and practice. Whether training had been received or not was an important factor affecting the knowledge, attitude, and practice of nurses, and the majority of participants had not received training. Nurses with lower levels of education had worse knowledge and practice, and contract nurses scored lower than staff nurses in terms of knowledge and attitude. CONCLUSIONS: The critical role of nurses in patient management places high demands on their knowledge, attitude, and practice. However, the present study demonstrated that the current knowledge, attitude, and practice of colorectal surgery nurses regarding the syndrome gave cause for concern, and whether training had been received was a key factor affecting these three aspects. Therefore, training is a key strategy to eliminate the gaps identified.

A study on autonomous suturing task assignment in robot-assisted minimally invasive surgery.

BACKGROUND: Sutures are a set of standard actions which are accomplished by multi-instruments, researchers studied the robot autonomy of suturing, which was based on movement planning completed by a single instrument, but did not consider the assignment of suturing tasks to instruments. METHOD: A method was proposed for the autonomous suturing task assignment to instruments, which built a comprehensive evaluation index under some constraint conditions to determine the optimal scheme of the suturing task assignment to instruments. RESULTS: An experiment of duodenal ulcer repair with a suturing operation was conducted under the guidance of a surgeon, and the results showed that the optimal scheme of the suturing task assignment was obtained by using the proposed method. CONCLUSIONS: The proposed method can be used for autonomous suturing task assignment, which is beneficial for improving the intelligence of robot operation.

Standard therapies: solutions for improving therapeutic effects of immune checkpoint inhibitors on colorectal cancer.

Immunotherapy using immune checkpoint inhibitors has opened a new era for cancer management. In colorectal cancer, patients with a phenotype of deficient mismatch repair or high microsatellite instability benefit from immunotherapy. However, the response of rest cases to immunotherapy alone is still poor. Nevertheless, preclinical data have revealed that either ionizing irradiation or chemotherapy can improve the tumoral immune milieu, because these approaches can induce immunogenic cell death among cancer cells. In this regard, combination use of standard therapy plus immunotherapy should be feasible. In this review, we will introduce the specific roles of standard therapies, including radiotherapy, chemotherapy, antiangiogenic and anti-EGFR therapy, in improving therapeutic effect of immune checkpoint inhibitors on colorectal cancer.

Spectral Mesh Segmentation via l0 Gradient Minimization.

Mesh segmentation is a process of partitioning a mesh model into meaningful parts - a fundamental problem in various disciplines. This paper introduces a novel mesh segmentation method inspired by sparsity pursuit. Based on the local geometric and topological information of a given mesh, we build a Laplacian matrix whose Fiedler vector is used to characterize the uniformity among elements of the same segment. By analyzing the Fiedler vector, we reformulate the mesh segmentation problem as a l0 gradient minimization problem. To solve this problem efficiently, we adopt a coarse-to-fine strategy. A fast heuristic algorithm is first devised to find a rational coarse segmentation, and then an optimization algorithm based on the alternating direction method of multiplier (ADMM) is proposed to refine the segment boundaries within their local regions. To extract the inherent hierarchical structure of the given mesh, our method performs segmentation in a recursive way. Experimental results demonstrate that the presented method outperforms the state-of-the-art segmentation methods when evaluated on the Princeton Segmentation Benchmark, the LIFL/LIRIS Segmentation Benchmark and a number of other complex meshes.

Pleomorphic rhabdomyosarcoma metastasis to small intestine causing intussusception: A case report.

RATIONALE: Pleomorphic rhabdomyosarcoma (PRMS) is a rare soft tissue malignancy which is frequently misdiagnosed and associated with metastasis to the lungs, lymph nodes, and bone marrow. Case studies are needed to improve the awareness of the disease and our understanding of it. PATIENT CONCERNS: In this study, we present a case of a 36-year-old man with a lesion on the right back shoulder. Lesion was confirmed by magnetic resonance imaging (MRI) around the right armpit, subscapularis, deltoid, and infraspinatus muscle, with oozing surrounding soft tissues. DIAGNOSIS: The tumor was diagnosed as PRMS which metastasized to the intestine, where it caused intussusception. INTERVENTIONS: The patient was treated by complete surgery in combination with neo-adjuvant chemotherapy including ifosfamide and epirubicin. OUTCOME: The patient remained alive 6 months after the treatment with no recurrence and metastasis. LESSON: PRMS can be aggressive, and surgical treatment in combination with multidrug chemotherapy can be used in the management.

The expression of serum sEGFR, sFlt-1, sEndoglin and PLGF in preeclampsia.

The objective of this study was to investigate soluble epidermal growth factor receptor (sEGFR), soluble vascular endothelial growth factor receptor 1 (sFlt-1), soluble endoglin (sEndoglin) and placenta growth factor (PLGF) concentrations in normotensive, preterm and term preeclamptic pregnancies' serum and thus to specify the clinical utility of these biochemical markers in monitoring severity and intrauterine growth retardation of preterm preeclampsia. 172 pregnant women were divided into the following groups: preterm preeclampsia, preterm control, term preeclampsia and term control. Preterm preeclampsia patients were stratified with severe feature (n = 50) and without severe feature (n = 22). sEGFR, sEndoglin and PLGF were assessed using Luminex multiplex immunoassay, whilesFlt-1 was assessed using ELISA. sEGFR was significantly lower in preterm preeclampsia than matched control (p < 0.001) and mildly lower in term preeclampsia than matched control (p < 0.01). On contrary, sFlt-1 was significantly higher in preterm preeclampsia than matched control (p < 0.001) and mildly higher in term preeclampsia than matched control (p < 0.01). sFlt-1, sFlt-1/sEGFR and sFlt-1/PLGF were positively correlated with the severity of preterm preeclampsia (P < 0.001, R value ≥ 0.6), especially sFlt-1/sEGFR had the highest R value (R value = 0.711) among them. Furthermore, sEndoglin and the ratio of sEndoglin/sEGFR were associated with neonatal birth weight small for gestational age (SGA, n = 25) in preterm preeclampsia group. CONCLUSIONS: The ratio of sFlt-1/sEGFR could be used as a novel candidate biochemical marker in monitoring the severity of preterm preeclampsia. sEndoglin and sEGFR may be involved in the pathogenesis of SGA in preterm preelampsia.

Serum protein marker panel for predicting preeclampsia.

BACKGROUND: Preeclampsia is a multi-system disorder in pregnancy which has no effective treatment. The diagnosis of preeclampsia is based on clinical presentation and routine laboratory tests. OBJECTIVE: This study aimed at identifying serum protein markers for diagnosis of preeclampsia and predicting its severe features. STUDY DESIGN: In total, 172 pregnant women were enrolled in this study including 110 subjects with preeclampsia and 62 normotensive subjects. Eleven serum proteins (VEGF, sFlt-1, sEndoglin, PlGF, sEGFR, prolactin, PTX3, PAI-1, NGAL, IL-27, COX-2) were assessed using Luminex multiplex immunoassay and ELISA. RESULTS: The levels of seven proteins (sFlt-1, VEGF, sEndoglin, sEGFR, PlGF, NGAL, COX-2) correlated with preeclampsia, and 4 proteins (VEGF, sEndoglin, PlGF, sEGFR) were identified as independent factors associated with preeclampsia. The levels of three proteins (sEndoglin, PTX3, sFlt-1) correlated with severe features of preeclampsia, and three variables (serum creatinine, platelet count and sEndoglin) were identified as independent factors in predicting severe features of preeclampsia. CONCLUSIONS: A combination of serum protein markers (VEGF, sEndoglin, PlGF, sEGFR) and clinical variables (serum creatinine, platelet count and sEndoglin) could be used as analytical tool in diagnosis of preeclampsia and its severe features, respectively. Serum sEGFR, a novel biomarker in preeclampsia, may be involved in the pathogenesis of preeclampsia.

Randomized controlled trial of uncut Roux-en-Y vs Billroth II reconstruction after distal gastrectomy for gastric cancer: Which technique is better for avoiding biliary reflux and gastritis?

AIM: To identify which technique is better for avoiding biliary reflux and gastritis between uncut Roux-en-Y and Billroth II reconstruction. METHODS: A total of 158 patients who underwent laparoscopy-assisted distal gastrectomy for gastric cancer at the First Hospital of Jilin University (Changchun, China) between February 2015 and February 2016 were randomized into two groups: uncut Roux-en-Y (group U) and Billroth II group (group B). Postoperative complications and relevant clinical data were compared between the two groups. RESULTS: According to the randomization table, each group included 79 patients. There was no significant difference in postoperative complications between groups U and B (7.6% vs 10.1%, P = 0.576). During the postoperative period, group U stomach pH values were lower than 7 and group B pH values were higher than 7. After 1 year of follow-up, group B presented a higher incidence of biliary reflux and alkaline gastritis. However, histopathology did not show a significant difference in gastritis diagnosis (P = 0.278), and the amount of residual food and gain of weight between the groups were also not significantly different. At 3 mo there was no evidence of partial recanalization of uncut staple line, but at 1 year the incidence was 13%. CONCLUSION: Compared with Billroth II reconstruction, uncut Roux-en-Y reconstruction is secure and feasible, and can effectively reduce the incidence of alkaline reflux, residual gastritis, and heartburn. Despite the incidence of recanalization, uncut Roux-en-Y should be widely applied.

Pose optimization and port placement for robot-assisted minimally invasive surgery in cholecystectomy.

BACKGROUND: Pose optimization and port placement are critical issues for preoperative preparation in robot-assisted minimally invasive surgery (RMIS), and affect the robot performance and surgery quality. METHODS: This paper proposes a method for pose optimization and port placement for RMIS in cholecystectomy that considers both the robot and surgery requirements. The robot pose optimization was divided into optimization of the positioning joint configuration and optimization of the end effector configuration. To determine the optimal location for the trocar port placement, the operational workspace was defined as the evaluation index. The port area was divided into many sub-areas, and that with the maximum operational workspace was selected as the location for the port placement. RESULTS: Considering the left robotic arm as an example, the location for the port placement and joints angles for robotic arm configuration were discussed and simulated using the proposed method. CONCLUSION: This research can provide guidelines for surgeons in preoperative preparation.

Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB, uPA activator and MMP9.

Curcumin, an active nontoxic ingredient of turmeric, possesses potent anti-inflammatory, antioxidant and anti-cancer properties; however, the molecular mechanisms of curcumin are not fully understood. The transcription factor nuclear factor-κB (NF-κB) is key in cellular processes, and the expression/activation of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) are crucial for cell invasion. The present study investigated the hypothesis that curcumin inhibits colon cancer cell invasion by modulating NF-κB-mediated expression and activation of uPA and MMP9. Human colon cancer SW480 and LoVo cells were treated with various concentrations of curcumin. Curcumin was demonstrated to dose-dependently inhibit the adhesion and proliferation ability of LoVo and SW480 cells using Transwell and MTT assays, respectively. In addition, curcumin activated 5' AMP-activated protein kinase (AMPK) and suppressed p65 NF-κB phosphorylation, as shown by western blot analysis. Compound C, a potent AMPK inhibitor, abolished curcumin-induced inhibition of NF-κB, uPA and MMP9, suggesting that AMPK activation is responsible for curcumin-mediated NF-κB, uPA and MMP9 inhibition. The binding activity of NF-κB to DNA was examined and western blotting and quantitative polymerase reaction was performed to detect the effect of curcumin on the expression of uPA and MMP9. The present results revealed that curcumin significantly decreased the expression of uPA and MMP9 and NF-κB DNA binding activity. Furthermore, curcumin decreased the level of the p65 subunit of NF-κB binding to the promoter of the gene encoding uPA and MMP9, which suppressed transcriptional activation of uPA and MMP9. Overall, the present data suggest that curcumin inhibits colon cancer cell invasion via AMPK activation and subsequent inhibition of p65 NF-κB, uPA and MMP9. The therapeutic potential of curcumin for colon cancer metastasis required additional study.

Serum biomarker panels for diagnosis of gastric cancer.

PURPOSE: Currently, serum biomarkers that are sufficiently sensitive and specific for early detection and risk classification of gastric adenocarcinomas are not known. In this study, ten serum markers were assessed using the Luminex system and enzyme-linked immunosorbent assay for the diagnosis of gastric cancer and analysis of the relation between prognosis and metastases. PATIENTS AND METHODS: A training set consisting of 228 gastric adenocarcinoma and 190 control samples was examined. A Luminex multiplex panel with nine biomarkers, consisting of three proteins discovered through our previous studies and six proteins previously reported to be cancer-associated, was constructed. One additional biomarker was detected using a commercial kit containing EDTA. Logistic regression, random forest (RF), and support vector machine (SVM) were used to identify the panel of discriminatory biomarkers in the training set. After selecting five proteins as candidate biomarkers, multivariate classification analyses were used to identify algorithms for diagnostic biomarker combinations. These algorithms were independently validated using a set of 57 gastric adenocarcinoma and 48 control samples. RESULTS: Serum pepsinogen I, serum pepsinogen II, A Disintegrin And Metalloproteinase domain-containing protein 8 (ADAM8), vascular endothelial growth factor (VEGF), and serum IgG to Helicobacter pylori were selected as classifiers in the three algorithms. These algorithms differentiated between the majority of gastric adenocarcinoma and control serum samples in the training/test set with high accuracy (RF 79.0%, SVM 83.8%, logistic regression 76.2%). These algorithms also differentiated the samples in the validation set (accuracy: RF 82.5%, SVM 86.1%, logistic regression 78.7%). CONCLUSION: A panel of combinatorial biomarkers comprising VEGF, ADAM8, IgG to H. pylori, serum pepsinogen I, and pepsinogen II were developed. The use of biomarkers is a less invasive method for the diagnosis of gastric adenocarcinoma. They may supplement clinical gastroscopic evaluation of symptomatic gastric cancer patients and enhance the diagnostic accuracy.

A novel immunotoxin - rCCK8PE38 targeting of CCK-R overexpressed colon cancers.

BACKGROUND: Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors. PURPOSE: Construct a new IT-targeting CCKR-overexpressing colon cancers. METHODS: To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice. RESULTS: The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18-26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8-3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice. CONCLUSIONS AND DISCUSSION: All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.

Sorcin Enhances Metastasis and Promotes Epithelial-to-Mesenchymal Transition of Colorectal Cancer.

Sorcin, a soluble resistance-related calcium-binding protein, belongs to the small penta-EF-hand family. Recent study reported that upregulation of sorcin correlated with metastasis and poor prognosis of colorectal cancer (CRC). In the present study, we explored the regulatory role of sorcin in CRC metastasis. To investigate the role of sorcin in CRC metastasis, sorcin overexpressed with empty vector as control in CRC cell line (HCT116). The effect of sorcin overexpression on cell migration and invasion was evaluated via wound healing and transwell assay, respectively. Sorcin-induced changes in EMT process were evaluated by estern blot. Furthermore, the role of PI3K/Akt in the regulatory effect of sorcin on cell migration and invasion, and EMT process was explored by suppressing Akt activity in sorcin-overexpressed HCT116 cells. Sorcin overexpression in HCT116 cells resulted in a significant increase in cell migration and invasion. Sorcin overexpression also markedly promoted the EMT process. More importantly, our results revealed that sorcin stimulated EMT process through activating PI3K/Akt signaling. In summary, this study indicated that the promoting effect of sorcin on CRC metastasis was, at least in part, through PI3K/Akt signaling. The findings in this study highlight the effectiveness and therapeutic potential to utilize sorcin-targeted strategies in the treatment of CRC.

Establishment of a three-step purification scheme for a recombinant protein rG17PE38 and its characteristics identification.

A protein with high purity has become an essential pre-requisite for investigating its bioactivity, molecular structure and characteristics. Therefore, the development of technologies for efficient purification of protein is urgently necessary. The objective of this study was to establish a purification protocol for a recombinant protein rG17PE38. Different forms of chromatography such as hydrophobic interaction and ion exchange chromatography were chosen as the core purification steps. The performance of each technique was optimized to meet the requirements and the purification steps were arranged in a logical way of facilitating to operate in next step. In addition, some characteristics of the protein such as stability, bioactivity and cellular location were determined. Finally, whether the protein could induce cell apoptosis was also explored. The results showed the protein purified via the suggested three-step purification scheme could obtain a purity of 95%, and its bioactivity in the form of IC50 was 17.6 ng/mL, furthermore it could keep stable at 4 °C for at least 10 days. The protein could bind on its target cell membrane specifically, and inducing cell apoptosis was demonstrated to be one of the cytotoxicity mechanisms of the protein. Results obtained in our study may provide useful information on strategies of protein purification and lay a substantial foundation for the followed animal or clinical experiments on rG17PE38.

Expression, purification and characterization of recombinant toxins consisting of truncated gastrin 17 and pseudomonas exotoxin.

Gastric cancer is a major cause of mortality and morbidity around world. However the effectiveness of the current approaches to the diagnosis and treatment of gastric cancer is limited. Recombinant targeted toxins may represent a novel direction of cancer therapy. In this study, we aimed to explore whether recombinant toxins fused with the truncated forms of G17 could target to kill cancer cells by recognizing CCK2R. Four recombinant Pseudomonas toxins PE38 fused with the forward or reverse truncated forms of G17 (G14 and G13) were successfully constructed, expressed, and purified. Their characteristics were further analyzed by SDS-PAGE, western blot and indirect immunofluorescence assay. The cytotoxicity assay demonstrated that only reversely fused recombinant toxins rG14PE38 and rG13PE38 exhibited certain toxicity on several cancer cell lines, and a competition assay indicated that the binding of the reverse gastrin-endotoxin to CCK2R (+) cells may be mediated by interaction between gastrin/gastrin-like and CCK2R.

rG17PE38, a novel immunotoxin target to gastric cancer with overexpressed CCK-2R.

BACKGROUND: Gastrin/cholecystokinin subtype 2 receptor (CCK2R) is overexpressed in several types of tumors. Gastrin-17 (G17) peptide has a high affinity with CCK2R. These characters suggest that G17 may be useful for target cancer therapy. PURPOSE: Construct a new immunotoxin (IT) targeting of CCK2R overexpressed gastric cancer. METHODS: Two ITs were generated using forward and reverse G17 peptides fused with PE38. To get a high yield, codon optimized gene and optimized fermentation parameters were used in large-scale protein expression. An immunoaffinity technique was introduced into pseudomonas exotoxin (PE)-derived IT purification procedure. G17 competition, GST pull-down and indirect immunoflourescence assays were carried out to confirm the interaction between rG17 and CCK2R. Then, several cytotoxic assays were carried out on 18 cell lines, and an in vivo antitumor activity experiment was tested in nude mice. RESULTS: The rG17PE38 showed specific cytotoxicity on three gastric cancer cells, while G17PE38 did not. After optimization, the expression level reached about 40% in medium deprived of NaCl. Next, 15-27.5 mg of pure rG17PE38 per 1 L of cultures was obtained. Results of G17 competition, GST pull-down and indirect immunoflourescence assays demonstrated that rG17 have a specific interact with CCK2R. Purified rG17PE38 showed high cytotoxicity on gastric cancer cell lines with the IC50 value of 0.6-4 ng·mL(-1). Treatment of nude mice inoculated with BGC-823 tumor xenografts with rG17PE38 efficiently inhibited tumor size. CONCLUSIONS AND DISCUSSION: The present study demonstrates that reversed G17 could be used as target moiety of PE-derived IT and the rG17PE38 could be developed as a new immunotherapy agent. Codon optimized gene could increase the rG17PE38 expression level in E. coli and furthermore NaCl inhibits the rG17PE38 expression in large scale. Meanwhile, our present study inducts an immunoaffinity method in the IT purification procedure, which could purify the PE-derived ITs in native form.