[Alpha-momordicin Regulates Hepatocyte Cytokine Expression].

OBJECTIVE: To investigate the regulation effect of α-momordicin (α-MMC) on the synthesis and secretion of cytokines in hepatocytes cells. METHODS: Hepatocytes L02 were treated with 189 µg/mL α-MMC with culture supernatant and lysate samples were harvested in different timepoint. Expressions of T-helper 17 (TH17) cytokine profile in samples were detected by the Bio-Plex 200 suspension chip assay system. RESULTS: Compared with 0 h, after the α-MMC treatment of L02 hepatocytes for 2 h, 4 h and 8 h, the intracellular synthesis of cytokines interleukin (IL)-1b, IL-6, IL-17A, IL-31, IL-33, soluble CD40 ligand (sCD40L), tumor necrosis factor-α (TNF-α) were all significantly decreased (P<0.05), and IL-6, IL-4, IL-17A, and sCD40L secreted into the extracellular fluid also decreased significantly (P<0.05). CONCLUSION: α-MMC can significantly inhibit the synthesis and secretion of cytokines such as IL-6, IL-17A and TNF-α in hepatocytes, which may become a side effect of its anti-tumor application.

PEGylation alleviates the non-specific toxicities of Alpha-Momorcharin and preserves its antitumor efficacy in vivo.

Alpha-Momorcharin (α-MMC) is a ribosome inactivating protein from Momordica charantia with anti-tumor activity. Previously, we had observed that modification of α-MMC with polyethylene glycol (PEG) could reduce toxicity, but it also reduces its anti-tumor activity in vitro. This study aims to investigate whether the metabolism-extended properties of α-MMC resulting from PEGylation could preserve its anti-tumor efficacy in vivo through pharmacokinetics and antitumor experiments. The pharmacokinetics experiments were conducted in rats using the TCA (Trichloroacetic Acid) method. Antitumor activity in vivo was investigated in murine mammary carcinoma (EMT-6) and human mammary carcinoma (MDA-MB-231) transplanted tumor mouse models. The results showed that PEGylation increased the plasma half-life of α-MMC in rats from 6.2-7.5 h to 52-87 h. When administered at 1 mg/kg, α-MMC-PEG and α-MMC showed similar anti-tumor activities in vivo, with a T/C% of 38.56% for α-MMC versus 35.43% for α-MMC-PEG in the EMT-6 tumor model and 36.30% for α-MMC versus 39.88% for α-MMC-PEG in the MDA-MB-231 tumor model (p > 0.05). Importantly, at the dose of 3 mg/kg, all the animals treated with α-MMC died while the animals treated with α-MMC-PEG exhibited only moderate toxic reactions, and α-MMC-PEG exhibited improved anti-tumor efficacy with a T/C% (relative tumor growth rate) of 25.18% and 21.07% in the EMT-6 and MDA-MB-231 tumor models, respectively. The present study demonstrates that PEGylation extends the half-life of α-MMC and alleviates non-specific toxicity, thereby preserving its antitumor efficacy in vivo, and a higher lever of dosage can be used to achieve better therapeutic efficacy.

[Effect of PEGylation of alpha-Momorcharin against its hepatotoxicity in rats].

OBJECTIVE: To explore the effect of PEGylation of alpha-Momorcharin (alpha-MMC), one of ribosome-inactivating proteins from bitter melon seed, against its hepatotoxicity in rats. METHODS: SD rats were randomized into NS group, alpha-MMC treated groups, and alpha-MMC-PEG treated groups. The doses of alpha-MMC and alpha-MMC-PEG were high, middle, and low dose (6.25, 2.08, 0.70 mg/kg). The rats were given different dose of alpha-MMC, or alpha-MMC-PEG via caudal vein every other day for consecutive 28 days and then left for 14 days recovery. The general condition of animals was observed, blood and liver samples were collected for liver function study and pathological examination on day 28 after initiation of administration and on day 14 after withdrawal. RESULTS: On day 28 after initiation of administration, the liver function damages were found in high-dose and middle-dose of alpha-MMC treated groups, such as the decreasing of ALB, increasing of GLB, A/G ratio decreasing and the dose-dependant increasing of AST, BIL and CHO. The pathological changes of hepatotoxicity were also observed in these two groups, including the massive hepatocyte, swelling degeneration, inflammatory cell infiltration, congestion and diffusive necrosis. However, the liver function and pathological changes in alpha-MMC-PEG treated groups were better than those in alpha-MMC treated groups. CONCLUSION: PEGylation could reduce the hepatotoxicity of alpha-MMC to rats.

PEGylation is effective in reducing immunogenicity, immunotoxicity, and hepatotoxicity of α-momorcharin in vivo.

BACKGROUND AND AIM: α-momorcharin (α-MMC), a type I ribosome-inactivating protein (RIP) from Momordica charantia, is well known for its antitumor and antivirus activities. However, the immunotoxicity and hepatotoxicity hampers its potential therapeutic usage. In order to reduce its toxicity, we had modified the α-MMC with polyethylene glycol (PEG), and detected the toxicity of the PEGylated α-MMC conjugates (α-MMC-PEG) in vivo. MATERIALS AND METHODS: After α-MMC purified from bitter melon seeds, α-MMC-PEG was constructed with a branched 20 kDa (mPEG) 2-Lys-NHS, the tests of immunogenicity, immunotoxicity, and general toxicity of α-MMC-PEG were conducted in guinea pig and rat. RESULTS: The titer of specific IgG in rats, immunized by α-MMC-PEG, were approximately one-third of those that by α-MMC, all the guinea pigs treated with α-MMC died of anaphylaxis shock within 5 min, while no animals treated with α-MMC-PEG died in the active systemic anaphylaxis (ASA) test. The passive cutaneous anaphylaxis (PCA) reaction of α-MMC-PEG challenge in rats was significantly smaller than that of the α-MMC. The liver damage was greatly released, such as the change of globulin (GLB), aspartate aminotransferase (AST), total bilirubin (TBIL) cholesterol (CHOL), albumin (ALB), and the degree of hepatocyte necrosis in repeated toxicity study. CONCLUSIONS: PEGylation is effective in reducing the immunogenicity, immunotoxicity, and hepatotoxicity of α-MMC in vivo.

[Study on the immunotoxicity of ribosome inhibiting protein of Momordica charantia].

OBJECTIVE: To separate and purify ribosome inhibiting protein (RIP) from Momordica charantia (bitter melon) seeds and to evaluate its acute toxicity and immunotoxicity in animal. METHODS: Ion exchange chromatography and gel filtration chromatography were applied in the separating and purifying of RIP from Momordica charantia seeds. Then the acute toxicity testing of RIP in mice was conducted to obtain its half lethal dose (LD50). Active systemic anaphylaxis(ASA)test in guinea pig and passive cutaneous anaphylaxis test (PCA) in rat were performed to evaluate its immunotoxicity. RESULTS: The LD50 (iv) in mice of RIP was 25.2 mg/kg in ASA, guinea pigs of the higher and lower RIP group all appeared stong allergic responses and most of them died quickly. In PCA, obvious blue dye in skin were observed in SD rats of the RIP group. CONCLUSION: RIP getting from Momordica charantia seeds had a relatively strong immunotoxicity in animals.

[Effects of KH901, a tumor-specific oncolytic recombinant adenovirus, on antitumor and expressing GM-CSF in xenograft tumor models].

OBJECTIVE: Conditionally replicating oncolytic adenovirus KH901 was engineered with a genetically modified telomerase reverse transcriptase promoter and a cDNA of human granulocyte macrophage colony stimulating factor (GM-CSF). The objective of this study was to evaluate the anti-tumor efficacy and the selective GM-CSF expression of KH901 in xenograft tumor models. METHODS: After intratumoral administration of KH901, the rates of Relative Tumor Growth (T/C%) and inhibition in Hep3B and LNcap xenograft models were measured for observing the KH901 antitumor efficacy. At various time points, the GM-CSF expression levels in tumor tissues and the blood of A549 xenograft model were determined by ELISA method. RESULTS: In both Hep3B and LNcap xenograft models, KH901 showed the significantly higher restraint tumor rates at high dose (3 X 10(10) VP, P<0. 05) compared to 5-FU or Cisplatin. Even at the low dose (3 X 10(8) VP), the KH901 antitumor effect was similar to 5-FU (P>0. 05). In A549 xenograft model, the level of GM-CSF was continuously elevated and the peak values were found on day 7 in the blood and on day 11 in the tumor tissues. Then GM-CSF expression gradually reduced in both blood and tumor tissues. CONCLUSION: KH901 can significantly inhibit the tumor growth in xenograft tumor model, and also express a high level of human GM-CSF in tumor tissue and release to circulating system to form a CM-CSF peak value in the blood.

[Tumor-selective replication, cytotoxicity and GM-CSF production of oncolytic recombinant adenovirus in KH901 injection].

OBJECTIVE: To study the tumor-selective replication, cytotoxicity and GM-CSF production of the recombinant virus in KH901 injection used to infect the cells cultured in vitro. METHODS: A panel of tumor and normal cells was infected with recombinant adenovirus in KH901 and wild-type adenovirus type 5 at a MOI of 2 PPC, the cells were harvested at 72 hours after infection and made a titer after three cycles of freeze/thaw; A panel of tumor and normal cells was infected with recombinant adenovirus KH901 at MOI of 1 or 10 PPC. For 24 hours after infection the medium was harvested to determine the biological activity of GM-CSF; A panel of tumor and normal cells was infected with KH901 of recombinant adenovirus and wild-type adenovirus type 5 at MOIs of 0, 0.1, 1, 10, 100, and 1000 PPC. At 7 days after infection, cell viability was determined by the MTT assay, and ECso was determined too. RESULTS: The data showed that wild-type adenovirus type 5 replicated efficiently in and killed both the tumor and normal cells, however, the recombinant adenovirus in KH901 replicated hugely in tumor cells [(2526.4+/-136.8)-(2796.6+/-104.6) TCID50/cell), and produced significant amount of GM-CSF [(1177. 793 +/-6.62)-(3924.497+/-17.79) IU/(10(6) cell x 24h)] and killed the tumor cells [EC50: (0.31+/-0.06)-(0.19+/- 0.01) pfu/cell] while was replicating poorly in non-permissive human normal cells [(56.8+/-9.2)-(90.1+/-14.4) TCID50/ cell], and producing very small amount of GM-CSF [(13.397+/-0.82) IU/(10(6)cell x 24 h)] and attenuating human primary cells killed [EC50: (92.33 +/- 9.12)-(121.20 +/- 19.94) pfu/cell], with which there was statistically a significant difference between wild-type adenovirus type 5 and recombinant adenovirus in KH901 (P<0.05). CONCLUSION: In vitro studies show that the tumor-selective replication, cytotoxicity, GM-CSF production of recombinant adenovirus lead the injection KH901 containing the recombinant adenovirus, as oncolytic agent, to have a potential utility for the treatment of solid tumors.