Therapeutic evaluation of monoclonal antibody-maytansinoid conjugate as a model of RON-targeted drug delivery for pancreatic cancer treatment.

Aberrant expression of the RON receptor tyrosine kinase, a member of the MET proto-oncogene family, contributes significantly to pancreatic cancer tumorigenesis and chemoresistance. Here we validate RON as a target for pancreatic cancer therapy using a novel anti-RON antibody Zt/g4-drug maytansinoid conjugates (Zt/g4-DM1) as a model for RON-targeted drug delivery to kill pancreatic cancer cells. In pancreatic cancer cell lines overexpressing RON, Zt/g4-DM1 rapidly induced receptor endocytosis, arrested cell cycle at G2/M phase, reduced cell viability, and subsequently caused massive cell death. These in vitro observations help to establish a correlation between the number of the cell surface RON receptors and the efficacy of Zt/g4-DM1 in reduction of cell viability. In mice, Zt/g4-DM1 pharmacokinetics in the linear dose range fitted into a two-compartment model with clearance in 0.21 ml/day/kg and terminal half-life at 6.05 days. These results helped to confirm a concentration-activity relationship for the BxPC-3 and other pancreatic cancer cell xenograft model with a tumoristatic dose at 3.02 mg/kg. Zt/g4-DM1 was effective in vivo against various xenograft PDAC growth but efficacy varied with individual cell lines. Combination of Zt/g4-DM1 with gemcitabine had a complete inhibition of xenograft pancreatic cancer growth. We conclude from these studies that increased RON expression in pancreatic cancer cells is a suitable targeting moiety for anti-RON ADC-directed drug delivery and anticancer therapy. Zt/g4-DM1 is highly effective alone or in combination with chemotherapeutics in inhibition of pancreatic cancer xenograft growth in preclinical models. These findings justify the use of humanized Zt/g4-DM1 for targeted pancreatic cancer therapy in the future.

Factors Associated with Relapse among Heroin Addicts: Evidence from a Two-Year Community-Based Follow-Up Study in China.

BACKGROUND: Many countries including China are facing a serious opiate dependence problem. Anti-drug work effectiveness was affected by the high relapse rate all over the world. This study aims to analyze the factors influencing heroin addict relapse, and to provide evidence for generating relapse prevention strategies. METHODS: A community-based follow-up study was conducted in China between October 2010 and September 2012. A total of 554 heroin addicts in accordance with the inclusion criteria from 81 streets in 12 districts of Shanghai, China were divided into 4 groups: group 1--daily dosage taken orally of 60 mL of methadone or under combined with psychological counseling and social supports (n = 130); group 2--daily dosage taken orally of over 60 mL of methadone combined with psychological counseling and social supports (n = 50); group 3--JTT (Jitai tablets) combined with psychological counseling and social supports (n = 206); group 4--JTT combined with social supports (n = 168). RESULTS: Log-rank test results showed that the cumulative relapse rate differences among four groups during the two-year follow-up period were not statistically significant (χ² = 5.889, p = 0.117). Multivariate Cox regression analysis results showed that only three independent variables were still statistically significant, including compliance with participation in psychological counseling (OR = 3.563, p = 0.000), the years of drug use (OR = 1.078, p = 0.001)and intervention model. CONCLUSIONS: Using the detoxification medications combined with appropriate psychological counseling and social support measures will help improve the effectiveness of relapse prevention, which is a kind of alternative community detoxification pattern. Appropriate and standard psychological counseling is very important for anti-drug treatment. The longer the drug addiction lasts, the longer the anti-drug treatment takes.

The effectiveness comparison of Jitai tablets versus methadone in community-based drug treatment: a 1-year follow-up study.

OBJECTIVE: The aim of the study was to compare the effectiveness of Jitai tablets (JTT) versus methadone in a community drug treatment program. METHODS: A cohort study was conducted with 386 eligible subjects from 7 districts to 65 communities in Shanghai. The subjects were placed into the JTT group (n=206) or the methadone group (n=180). The data were collected at 8-, 26- and 52-week follow-ups. RESULTS: The retention rates of the methadone group at the 8-, 26-, and 52-week follow-ups were 97.78%, 91.67%, and 85.00%, respectively. The retention rates of the JTT group at these follow-ups were 90.78%, 83.50%, and 74.27%, respectively. A Chi-square test indicated a significant difference, and the P values were 0.0037, 0.0161, and 0.0095 for each follow-up. The relapse rates for the JTT group were 3.88%, 6.31% and 11.17% for each follow-up, and those for the methadone group were 1.11%, 2.78%, and 7.78% for each follow-up. The Chi-square test indicated no significance, and the P values were 0.1128, 0.1005 and 0.2594. A survival analysis indicated that the relapse survival curve had no significant difference between the two groups (log-rank test, P=0.188). CONCLUSION: Methadone and JTT combined with psychological intervention and social support provided effective maintenance treatment and relapse prevention in a community drug treatment program. The retention rate in the methadone group was higher, but the JTT group had the same relapse prevention as the methadone group. JTT can be recommended to clinical doctors and drug addicts.

Oncogenic variant RON160 expression in breast cancer and its potential as a therapeutic target by small molecule tyrosine kinase inhibitor.

Aberrant expression of the RON receptor tyrosine kinase contributes to breast cancer malignancy. Although clinical trials of RON targeting are underway, the intriguing issue is the diversity of RON expression as evident by cancer cells expressing different variants including oncogenic RON160. The current study determines aberrant RON160 expression in breast cancer and its potential as a target for breast cancer therapy. Using mouse monoclonal antibody Zt/h12 in immunohistochemical staining of breast cancer tissue microarray, we observed that RON160 was expressed in high frequency in primary invasive ductal (77.2%, 61/79 cases), lobular (42.5%, 34/80 cases), and lymph node-involved (63.9%, 26/36 cases) breast cancer samples. Moreover, RON160 overexpression was predominantly observed in invasive ductal (26.6%, 21/79 cases) and lymph node-involved (33.3%, 12/36) cases. Among a panel of breast cancer cell lines analyzed, Du4475 cells naturally expressed RON160. Silencing RON160 expression by siRNA reduced Du4475 cell viability. Inhibition of RON160 signaling by tyrosine kinase inhibitor PHA665752 also suppressed Du4475 cell anchorage-independent growth and induced apoptotic cell death. Studies in vivo revealed that PHA665752 inhibited 3T3- RON160 and Du4475 cell-mediated tumor growth in mouse mammary fat pad. A 60% reduction in tumor volume compared to controls was achieved after a 13-day treatment. We conclude from these studies that RON160 is highly expressed in breast cancer and its signaling is integrated into cellular signaling network for tumor cell growth and survival. Experimental treatment by PHA665752 in Du4475 breast cancer xenograft model highlights the significance of RON160 as a drug target in molecular-targeted breast cancer therapy.

Hair analysis, a reliable and non-invasive method to evaluate the contamination by clenbuterol.

The illegal use of clenbuterol has been an increasingly serious issue in today's livestock products industry. It becomes an important project to develop a reliable approach to detect its content in food animals. A simple and sensitive LC-MS/MS method was developed to detect clenbuterol residue in hair, with the low limit of quantitation (LLOQ) about 0.5ng/g. Hogs fed with 340µg/day of clenbuterol for 2 weeks were found a high clenbuterol residue in their hair approximately at 1-2 months after withdrawal. There remained 3.31ng/g clenbuterol in hog hair approximately 5 months after the last administration, focused on the tip of the hair (mainly in hogs with dark hair). An extensive contamination was observed in twenty investigated market hogs whose dark hair obviously had a higher clenbuterol residue than the light ones (p=0.017, t test). Volunteers (60.3 percent) from Xuhui district (Shanghai) were found to have a detectable amount of clenbuterol in their hair (>0.5ng/g). In conclusion, hair residue detection is a reliable method to evaluate the clenbuterol contamination in animals and humans. Meat supply in the Xuhui district might have serious potential safety risks which should be further investigated and discussed to determine the safety range of clenbuterol residue.

The monoclonal antibody Zt/f2 targeting RON receptor tyrosine kinase as potential therapeutics against tumor growth-mediated by colon cancer cells.

BACKGROUND: Overexpression of the RON receptor tyrosine kinase contributes to epithelial cell transformation, malignant progression, and acquired drug resistance. RON also has been considered as a potential target for therapeutic intervention. This study determines biochemical features and inhibitory activity of a mouse monoclonal antibody (mAb) Zt/f2 in experimental cancer therapy. RESULTS: Zt/f2 is a mouse IgG2a mAb that is highly specific and sensitive to human RON and its oncogenic variants such as RON160 (ED(50) = 2.3 nmol/L). Receptor binding studies revealed that Zt/f2 interacts with an epitope(s) located in a 49 amino acid sequence coded by exon 11 in the RON ß-chain extracellular sequences. This sequence is critical in regulating RON maturation and phosphorylation. Zt/f2 did not compete with ligand macrophage-stimulating protein for binding to RON; however, its engagement effectively induced RON internalization, which diminishes RON expression and impairs downstream signaling activation. These biochemical features provide the cellular basis for the use of Zt/f2 to inhibit tumor growth in animal model. Repeated administration of Zt/f2 as a single agent into Balb/c mice results in partial inhibition of tumor growth caused by transformed NIH-3T3 cells expressing oncogenic RON160. Colon cancer HT-29 cell-mediated tumor growth in athymic nude mice also was attenuated following Zt/f2 treatment. In both cases, ~50% inhibition of tumor growth as measured by tumor volume was achieved. Moreover, Zt/f2 in combination with 5-fluorouracil showed an enhanced inhibition effect of ~80% on HT-29 cell-mediated tumor growth in vivo. CONCLUSIONS: Zt/f2 is a potential therapeutic mAb capable of inhibiting RON-mediated oncogenesis by colon cancer cells in animal models. The inhibitory effect of Zt/f2 in vivo in combination with chemoagent 5-fluorouracil could represent a novel strategy for future colon cancer therapy.

Ribosomal protein S6 kinase (RSK)-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein.

BACKGROUND: Epithelial to mesenchymal transition (EMT) occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP) has been implicated in cellular EMT program; however, the major signaling determinant(s) responsible for MSP-induced EMT is unknown. RESULTS: The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF)-ß1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. CONCLUSIONS: MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.