Recombinant Mutant Human Tumor Necrosis Research Articles

Thoracic perfusion of recombinant mutant human tumor necrosis factor (rmhTNF) can be considered as a good adjunct in the treatment of malignant pleural effusion caused by lung cancer

BackgroundTumor necrosis factor (TNF) has been investigated to be correlated with the occurrence and progression of lung cancer. This investigation was to assess the efficacy and safety of recombinant mutant human tumor necrosis factor (rmhTNF) for controlling malignant pleural effusion (MPE) through thoracic perfusion.MethodsThrough searching from MEDLINE, Web of Science, EMBASE, Cochrance Library, OVID and China National Knowledge Infrastructure (CNKI), a total of 12 studies with 694 patients were included in this study. A series of meta-analysis methods were used to analyze the extracted data.ResultsThoracic perfusion of rmhTNF combined with cisplatin promoted the objective response rate (ORR) (P < 0.001; odds ratio = 4.49) and the quality of life (QOL) of patients with MPE (P < 0.001; odds ratio = 10.33), as compared with cisplatin alone. Although the participation of rmhTNF increased the incidence of fever (P < 0.001), it seemed to relieve the adverse reactions in the digestive tract (P = 0.017).ConclusionsThoracic perfusion of rmhTNF contributes to the treatment of MPE and improves the QOL of MPE patients.

Hyperthermic intraperitoneal chemotherapy with recombinant mutant human TNF-α and raltitrexed in mice with colorectal-peritoneal carcinomatosis.

Peritoneum is one of the most common metastatic sites of colorectal cancer (CRC). It has been reported that cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) prolongs the lifespan of patients with peritoneal carcinomatosis of colorectal origin (CRC-PC), while the drugs used for HIPEC are limited. We investigated the application of recombinant mutant human tumor necrosis factor-α (rmhTNF) combined with raltitrexed in the HIPEC treatment in a mice model with CRC-PC. In vitro, we detected the cytotoxicity and apoptosis of human colorectal cancer cells by 3–(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, Western blot, and TdT-mediated dUTP Nick End Labeling (TUNEL) assay. In vivo, we established xenograft models of CRC-PC and assessed the antitumor effect by in vivo imaging, peritoneal cancer index scoring, and TUNEL assay. The results showed that the combination of rmhTNF and raltitrexed under hyperthermia with a temperature of 42°C inhibited the growth of colorectal cancer cells significantly in vitro, and after HIPEC treatments with rmhTNF and raltitrexed, peritoneal tumor growth was prohibited in vivo. Our findings about the efficacy of rmhTNF and raltitrexed used for HIPEC to treat CRC-PC will provide experimental data and basis for their potential clinical application. Impact statement Colorectal peritoneal carcinomatosis exhibits poor prognosis and presents a treatment challenge. At present, the main treatment is surgery, supplemented by hyperthermic intraperitoneal chemotherapy (HIPEC), but the drugs used for HIPEC are limited. Our study found that the combination of recombinant mutant human TNF-α (rmhTNF) and raltitrexed (RTX) under hyperthermia with a temperature of 42°C had antitumor effect both in vitro and vivo. The findings will provide experimental data and basis for the potential clinical application of rmhTNF and RTX, which might offer patients a new choice of therapeutic drugs.

Synergistic effects of rmhTRAIL and 17-AAG on the proliferation and apoptosis of multiple myeloma cells

ABSTRACT Objective: This study aimed to investigate synergistic effects of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) and heat-shock protein 90 (HSP90) inhibitor (geldanamycin derivative 17 -allylamino- 17-demethoxy -geldanamycin, 17-AAG) on the proliferation and apoptosis of multiple myeloma (MM) cells. Methods: MTT assays evaluated inhibitory effects of rmhTRAIL and 17-AAG in different concentrations and treatment durations on the proliferation of RPMI8226 and U266 cells. The half maximal inhibitory concentration was calculated using OriginPro7.5. Synergistic effects of rmhTRAIL and 17-AAG on apoptosis of MM cells were detected using flow cytometry at 24 and 48 h post-treatment. To evaluate synergistic effects of rmhTRAIL and 17-AAG, the Q-value was calculated using King's formula. Results: rmhTRAIL exhibited significant inhibitory effects on the proliferation of RPMI8226 cells in a dose- and time-dependent manner (>50%), whereas U266 cells were not sensitive to rmhTRAIL (<50%). 17-AAG inhibited the proliferation of RPMI8226 and U266 cells in a dose-dependent manner (>80%). Significant synergistic effects of rmhTRAIL and 17-AAG on the proliferation of RPMI8226 cells were revealed (Q-value > 1.15), whereas synergistic effects were not evident on the proliferation of U266 cells (Q-value < 1.15). rmhTRAIL and 17-AAG exhibited significant synergistic effects on apoptosis of RPMI8226 and U266 cells (Q-value > 1.15). Conclusion: The combined application of rmhTRAIL and 17-AAG revealed favorable synergistic effects in the treatment of MM.

Target and resistance-related proteins of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand on myeloma cell lines.

Recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) has become a potential therapeutic drug for multiple myeloma (MM). However, the exact targets and resistance mechanisms of rmhTRAIL on MM cells remain to be elucidated. The present study aimed to investigate the target and resistance-related proteins of rmhTRAIL on myeloma cell lines. A TRAIL-sensitive myeloma cell line, RPMI 8226, and a TRAIL-resistance one, U266, were chosen and the differentially expressed proteins between the two cell lines were analyzed prior and subsequent to rmhTRAIL administration by a liquid chromatography-tandem mass spectrometry method. The results showed that following TRAIL treatment, 6 apoptosis-related proteins, calpain small subunit 1 (CPNS1), peflin (PEF1), B-cell receptor-associated protein 31 (BAP31), apoptosis-associated speck-like protein containing CARD (ASC), BAG family molecular chaperone regulator 2 (BAG2) and chromobox protein homolog 3 (CBX3), were upregulated in RPMI 8226 cells while no change was identified in the U266 cells. Furthermore, small ubiquitin-related modifier 1 and several other ubiquitin proteasome pathway (UPP)-related proteins expressed higher levels in TRAIL-resistant cells U266 compared to the RPMI-8226 cells prior and subsequent to rmhTRAIL treatment. These results suggested that CPNS1, PEF1, BAP31, ASC, BAG2 and CBX3 were possibly target proteins of rmhTRAIL on RPMI 8226 cells, while UPP may have a vital role in mediating TRAIL-resistance in U266 cells.

RmhTNF-α combined with cisplatin inhibits proliferation of A549 cell line in vitro.

To explore the inhibitory effect of recombinant mutant human tumor necrosis factor-Α (rmhTNF-Α) in combination with cisplatin on human lung adenocarcinoma cell line A549. Human lung adenocarcinoma cell line A549 was treated with varying concentrations of rmhTNF-Α (0.38, 0.75, 1.50, 6.00 and 12.00 IU/ml) or cisplatin (3.91, 7.81, 15.63, 31.25 and 62.50 Μg/ml) for 24 hours. Viable cell number was analyzed by using crystal violet staining. The inhibitory rates of A549 cells growth by the two drugs were calculated. For analyzing whether there was a synergistic effect of rmhTNF-Α with cisplatin, A549 cells were treated with 0.75 IU/ml rmhTNF-Α and increased concentrations of cisplatin. rmhTNF-Α or cisplatin inhibited the growth of A549 cell lines in a dose-dependent manner. The inhibitory effect of rmhTNF-Α combined with cisplatin was significantly greater than cisplatin alone at the same concentration (all P<0.01). rmhTNF-Α combined with cisplatin might have synergistic inhibitory effect on human lung adenocarcinoma cell line A549.

A novel peptide (GX1) homing to gastric cancer vasculature inhibits angiogenesis and cooperates with TNF alpha in anti-tumor therapy

BackgroundThe discovery of the importance of angiogenesis in tumor growth has emphasized the need to find specific vascular targets for tumor-targeted therapies. Previously, using phage display technology, we identified the peptide GX1 as having the ability to target the gastric cancer vasculature. The present study investigated the bioactivities of GX1, as well as its potential ability to cooperate with recombinant mutant human tumor necrosis factor alpha (rmhTNFα), in gastric cancer therapy.ResultsTetrazolium salt (MTT) assay showed that GX1 could inhibit cell proliferation of both human umbilical vein endothelial cells (HUVEC) (44%) and HUVEC with tumor endothelium characteristics, generated by culturing in tumor-conditioned medium (co-HUVEC) (62%). Flow-cytometry (FCM) and western blot assays showed that GX1 increased the rate of apoptosis from 11% to 31% (p < 0.01) by up-regulating caspase 3 expression level. A chorioallantoic membrane assay indicated that GX1 could suppress neovascularization in vivo, with the microvessel count decreasing from 21 to 11 (p < 0.05). When GX1 was fused to rmhTNFα, GX1-rmhTNFα selectively concentrated in the gastric cancer vasculature, as shown by enzyme-linked immunosorbent assay, immunofluorescence and emission-computed tomography. In vitro MTT and FCM assays showed that, compared to rmhTNFα alone, GX1-rmhTNFα was more effective at suppressing co-HUVEC proliferation (45% vs. 61%, p < 0.05) and inducing apoptosis (11% vs. 23%, p < 0.05). In a tumor formation test, GX1-rmhTNFα more effectively inhibited tumor growth than rmhTNFα (tumor volume: 271 mm3 vs. 134 mm3, p < 0.05), with less systemic toxicity as measured by body weight (20.57 g vs. 19.30 g, p < 0.05). These therapeutic effects may be mediated by selectively enhanced tumor vascular permeability, as indicated by Evan's blue assay.ConclusionGX1 had both homing activity and the ability to inhibit vascular endothelial cell proliferation in vitro and neovascularization in vivo. Furthermore, when GX1 was conjugated to rmhTNFα, the fusion protein was selectively delivered to targeted tumor sites, significantly improving the anti-tumor activity of rmhTNFα and decreasing systemic toxicity. These results demonstrate the potential of GX1 as a homing peptide in vascular targeted therapy for gastric cancer.

Expression, Purification, and Characterization of Recombinant Protein GX1-rmhTNFα

A phage-displayed peptide CGNSNPKSC (GX1) was obtained previously in our lab, which could specifically bind to the vasculature of human gastric cancer. GX1-rmhTNFalpha was a fusion protein of GX1 and recombinant mutant human tumor necrosis factor alpha (rmhTNFalpha), which was designed by us with the expectation of enhancing selectivity of rmhTNFalpha. The DNA fragment encoding GX1 was cloned into the vector pBV220 with rmhTNFalpha between the EcoRI site and the BamHI site, and then expressed in Escherichia coli DH5alpha by temperature induction. Subsequently, E. coli DH5alpha was lysed, and the GX1-rmhTNFalpha protein was found in both soluble form and inclusion bodies. The protein was fractionated with ammonium sulfate deposition from 30% to 60%, and purified by cation and anion exchange chromatography using SP Sepharose Fast Flow column and Q Sepharose Fast Flow column. The purity of protein was then identified by SDS-PAGE and HPLC. Subsequent studies showed that GX1-rmhTNFalpha had high bioactivity of 5.65 x 10(8) IU/ml, which was similar with natural human TNFalpha and could reach the tumor site relatively faster than rmhTNFalpha.

Controlling malignant pericardial effusion by intrapericardial administration of recombinant mutant human tumor necrosis factor in patients with carcinoma

Objective To evaluate the therapeutic efficacy of injecting recombinant mutant human tumor necrosis factor (rmhTNF) into pericardial cavity of carcinoma patients with malignant pericardial effusion.

The role of the K+ channel in the inhibition of the human lung adenocarcinoma cell proliferation by rmhTNF

With the development of patch clamp and molecular biology technique, and the application of them in the investigation of tumor cellular membrane ion channnel, the ion channel is becoming the hot spot of the tumor base research gradually. The aim of this study is to investigate the electrophysiological properties of human lung adenocarcinoma cell line A-549 and the role of K+ channel in inhibition of cell proliferation by the recombinant mutant human tumor necrosis factor (rmhTNF). Ionic currents were recorded using the whole-cell patch clamp recording technique. The proliferation activity of A-549 cells was measured by MTT assay. The cell cycle and apoptosis rates of the carcinoma cells were measured by flow cytometric analysis (FCM). Whole-cell patch clamp recording revealed a voltage-gated K+ current in A-549 cells, which could be blocked by the K+ channel blocker, TEA and CsCl. The amplitude of K+ current was markedly diminished in all cells incubated with different concentration of rmhTNF (P < 0.01). Obvious inhibitive effect of rmhTNF on proliferation of the cells was found in vitro in a dose-dependent manner (P < 0.01), the maximal inhibitory rate was 38.68% when the concentration was 400U/mL. The rmhTNF inhibited the cell cycle shifting from G1 phase to S phase and promoted apoptosis as determined by FCM analysis. The proportion of G1 cells increased from 53.02% to 72.93%, and the apoptosis rate increased from 2.08% to 8.68%. The difference were significant between the control and the high concentration groups ( 200U/mL and 400U/mL) (P < 0.01). rmhTNF exerts its cytotoxic effects on A-549 cells through inhibiting cell cycle shifting and inducing apoptosis. The K+ channels on the A-549 cell membrane can be blocked by rmhTNF partly, and the effect of inhibiting proliferation and activating apoptosis on A-549 cells is a result of depression of the K+ channel.

Influence of rmhTNF on the chemotherapy treatment of small cell lung cancer

The efficacy and safety of the recombinant mutant human tumor necrosis factor (rmhTNF) combined with chemotherapy vs chemotherapy alone in the treatment of patients with small cell lung cancer (SCLC) were evaluated in this study. The selected 37 patients with SCLC were divided into experimental group (n =18) and control group (n = 19). Both groups were subjected to EP regimen. While in the experimental group, a regimen of 4 x 10(6) U/m2 rmhTNF intramuscular injection was given once a day from the 1st to 7th day and 11th to 17th day on the chemotherapy cycle. Twenty-one days were as a chemotherapy cycle and all patients received treatment with 2 cycles. The response rate was 83.3% (15/18) in the experimental group and 63.2% (12/19) in the control group respectively (P < 0.05). The KPS score after treatment was 78.4 +/- 9.6 in the experimental group and 71.2 +/- 9.7 in the control group with the difference being significant (P < 0.05). No severe adverse effects occurred in the two groups. It was concluded that the curative effectiveness of the rmhTNF combined with chemotherapy in the treatment of SCLC was more satisfactory than chemotherapy alone. The former could obviously improve the quality of life of the patients with SCLC.

Antitumor activity of a novel recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand1

To investigate the antitumor activity and safety of a novel recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmh TRAIL). Antitumor activity of rmh TRAIL was evaluated by using several tumor cell lines by MTT assay in vitro, and by using a mouse xenograft model in vivo. rmh TRAIL-induced apoptosis in tumor cells was detected by cell death enzyme-linked immunosorbent assay (ELISA), TdT-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry. The safety of rmh TRAIL was also evaluated in several normal human cell lines. At the concentration of 0.32-1 000 ng/mL, rmh TRAIL remarkably inhibited the proliferation of 5 tumor cell lines from lung, colon, and breast cancer compared with wild type (wt TRAIL) in vitro, whereas at the concentration of 1 ng/mL-10 microg/mL, rmh TRAIL showed no or mild cytotoxicity in the normal cell lines. rmh TRAIL (3, 15 mg/kg, ip, once daily for 10 d) exerted a significant inhibition on the growth of xenograft tumor NCI-H460 in nude mice compared with the saline group (P<0.01), and was more potent than wt TRAIL, a positive control. The apoptosis of NCI-H460 cells was markedly induced in a concentration-dependent and time-dependent manner after rmh TRAIL treatment. The percentage of apoptotic cells induced by rmh TRAIL in NCI-H460 cells was significantly higher than that by wt TRAIL. rmh TRAIL provided potent antitumor activity in vivo and in vitro, whereas most normal human cells were resistant to rmh TRAIL. The results suggested that rmh TRAIL might be a useful anticancer agent in future.

Recombination Mutant Human Tumor Necrosis Factor Combined with Chemotherapy in the Treatment of Advanced Cancer

Objective: Past studies showed that tumor necrosis factor (TNF) assisted anti-tumor treatment and intensified the sensitivity of chemotherapy. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. The objective of present study is to evaluate the therapeutic effects and adverse reactions of recombinant mutant human tumor necrosis factor (rmhTNF) combined with chemotherapy in patients with advanced malignant tumor. Methods: 105 patients with advanced malignant tumor were randomly divided into trial group, 69 patients, and control group, 36 patients. rmhTNF was injected intramuscularly to the trial group at a dose of 4×106 U/m2, from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP for patients with the NSCLC; FAM for patients with gastric cancer; FC for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. The control group was given only the same chemotherapy as the trial group. Results: In the trial group there was 1 CR case and 12 PR cases, and the response rate was 13/69 (18.84%); in the control group 1 PR case, the response rate 1/36 (2.78%). The response rate in the trial group was significantly higher than that in the control group (P=0.022). The response rate for NSCLC in the trial group was 8/17 (47.06%), and 1/6 (16.67%) in the control group. The response rates for gastric cancer and colorectal cancer in the trial groups also were higher than those in the control groups. After the treatment the KPS was 89.00±9.92 in the trial group, and 84.17±8.84 in the control group, with a significant difference between the two groups (P=0.028). The adverse reactions of rmhTNF injection included: pain in the injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia, and cold-like symptoms. All these adverse reactions were mild and bearable. Conclusion: The administration of rmhTNF in combination with general chemotherapy is an effective and secure means in treating advanced malignant tumor.

The effect of chemotherapy combined with recombination mutant human tumor necrosis factor on advanced cancer

BackgroundPast studies suggested that tumor necrosis factor (TNF) assisted anti-tumor treatment and intensified the sensitivity of chemotherapy. However its clinical application has been curbed because of its low purity, high dosage, and strong toxicity. This research, through perspective random clinical control experiment, observed the therapeutic effect of the treatment of late malignant tumor through the injection of recombinant mutant human tumor necrosis factor (rmhTNF) combined with general chemotherapy and its adverse reactions.Methods105 patients with advanced malignant tumor were randomly divided into trial group, 69 patients, and control group, 36 patients. Injection of rmhTNF 4 × 106u/m2 was given to the trial group, from the 1st to 7th days, the 11th to 17th days combined with chemotherapy course. The chemotherapy plan was as follows: CAP for patients with the NSCLC; FAM for patients with gastric cancer; FC for patients with colorectal cancer. One treatment cycle lasted for 21 days and two cycles were scheduled. The control group was given only the same chemotherapy as the trial group.ResultsIn the trial group there was 1 CR case and 12 PR cases, and the response rate is 13/69 (18.84%); in the control group 1 PR case, the response rate 1/36 (2.78%). The response rate of the trial group was significantly higher than that of the control group (P = 0.022). The response rate for NSCLC in the trial group was 8/17 (47.06%), and 1/6 (16.67%) in the control group. The response rates for gastric cancer and colorectal cancer in the trial groups also were higher than those of the control groups. After the treatment the KPS is 89.00 ± 9.92 in the trial group, and 84.17 ± 8.84 in the control group, with a significant difference between the two groups (P = 0.028). The adverse reactions of rmhTNF injection included: pain in the injection area, chill, hardening and swelling and redness in the injection area, fever, ostealgia and myosalgia, and cold-like symptoms. All these adverse reactions were mild and bearable.ConclusionsThe administration of rmhTNF injection in combination with general chemotherapy is an effective and secure means in treating advanced malignant tumor.

A multicenter randomized phase III trial of domestic product of rmhTNF in the treatment of non-small cell lung cancer

To evaluate and compare the effects and toxicity of the domestic product of recombinant mutant human tumor necrosis factor (rmhTNF) combined with chemotherapy and chemotherapy alone in the treatment of patients with non-small cell lung cancer (NSCLC). Two hundred patients with NSCLC in multicenter were randomly devided into trial group (150 cases) and control group (50 cases). Chemotherapy with CAP regimen was given to the patients. Meanwhile, rmhTNF injection of 4×10⁶U/m² was also given from the 1st to 7th days, the 11th to 17th days on the chemotherapy cycle in the trial group. The control patients received chemotherapy alone. Twenty-one days were as a cycle, 2 cycles were given to each patient. The chemotherapeutic effects and toxicity were observed and compared between the two groups after the therapy. of the 200 patients, 5 cases in the trial group and 3 cases in the control group were out of the trial because of economy. The other 192 cases (145 cases in the trial group and 47 cases in the control group) could be analyzed and evaluated the clinical effects and toxicity. The response rate of chemotherapy was 46.90% (68/145) in the trial group and 17.02% (8/47) in the control group respectively ( P =0.001). The KPS scores was 86.02±9.74 in the trial group, and 80.14±9.10 in the control group ( P =0.025). No significant difference of degree III+IV toxicity was observed between the two groups ( P > 0.05). The side effects related to rmhTNF included slight fever, cold-like symptoms, pain and red and swelling in the injection site. All of them were mild and didn't need any treatment and disappeared after the therapy. There were no severe abnormality of liver and kidney function and ECG in both groups. The results demonstrate that the effects of domestic rmhTNF combined with chemotherapy are remarkably higher than that of chemotherapy alone in the treatment of NSCLC. rmhTNF can increase the sensitivity to chemotherapy and improve the quality of life of the patients with slight toxicity. Hence rmhTNF is worth expanding clinical use.