EpCAM expression in esophageal cancer and its correlation with immunotherapy of solitomab.

BACKGROUND: Recurrence of esophageal cancer (EC) after chemotherapy may mainly be explained by the existence of chemotherapy-resistant cells, and an effective drug against chemotherapy-resistant cells is highly sought. The aim of this study was to investigate the cytotoxicity of bispecific antibody solitomab combined with γ δ T cells on Eca109 cell spheres. METHODS: We cultured Eca109 cell spheres in serum-free medium, and the morphological differences between wild-type Eca109 cells and Eca109 cell spheres were compared by microscope and flow cytometry. Different concentrations of nanoparticle albumin-bound paclitaxel (Nab-PTX) and cisplatin were used to treat the two groups of cells and compare their drug resistance. Flow cytometry was then used to detect the expression level of epithelial cell adhesion molecule (EpCAM) and the cytotoxicity of γ δ T cells combined with bispecific antibody solitomab on the two groups. RESULTS: Flow cytometry analysis showed that Eca109 cell spheres were smaller in size and had less cytoplasmic granules and CCK-8 assay showed that the viability of Eca109 cell spheres treated with different concentrations of Nab-PTX and cisplatin was significantly higher than that of wild-type Eca109 cells (P<0.05). Flow cytometry also showed that the expression level of EpCAM on Eca109 cell spheres was higher than that of wild-type Eca109 cells. Co-culture experiment showed that there was no significant difference in the cytotoxicity of γ δ T cells to wild-type Eca109 cells and Eca109 cell spheres without solitomab. However, after adding solitomab, the cytotoxicity of γ δ T cells to Eca109 cell spheres was significantly higher than that of wild-type Eca109 cells (P<0.05). CONCLUSIONS: EC Eca109 cell spheres have strong stem cell characteristics such as multidrug resistance and may contain a high proportion of EC stem cells. Further, EC Eca109 cell spheres have a high expression level of EpCAM, and EpCAM may be one of the markers of EC stem cells. Therefore, EpCAM could be used as a potential molecular target of immunotherapy for EC, and solitomab may become an effective immunotherapeutic drug for chemotherapy-resistant EC cells.

[Study on Molecular Biology of Rare Two A307 Phenotypes].

OBJECTIVE: To identify the genotypes of the 2 blood samples whose serological typing were difficult by DNA sequencing analysis, and to investigate the molecular genetic basis of their genotypes. METHODS: The 2 blood samples were preliminary genotyped by PCR-SSP. The complete exon 6 and 7 in the ABO genes were amplified by PCR and the PCR products were directly sequenced and clonal sequenced in order to identify the genotypes. RESULTS: The forward typing showed that both samples were weak A, while the reverse typing showed that the samples contained anti-A1. They were preliminarily genotyped as A/O1. RESULTS: The sequencing analysis showed that the 2 samples contained the nt467C>T and nt745C>T mutation in the A allele, which resulted in an amino acid change from Proline (Pro) to Leucine (Leu) at codon 156 and also from Arginine (Arg) to Tryptophan (Trp) at codon 249. CONCLUSION: Through serology results and sequencing analysis, the 2 samples are identified as rare A307 phenotypes.

[Preparation of hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata and study on its in vitro release mechanism].

In this study, hydrophilic matrix sustained release tablets of total lactones from Andrographis paniculata were prepared and the in vitro release behavior were also evaluated. The optimal prescription was achieved by studying the main factor of the type and amount of hydroxypropyl methylcellulose (HPMC) using single factor test and evaluating through cumulative release of three lactones. No burst drug release from the obtained matrix tablets was observed. Drug release sustained to 14 h. The release mechanism of three lactones from A. paniculata was accessed by zero-order, first-order, Higuchi and Peppas equation. The release behavior of total lactones from A. paniculata was better agreed with Higuchi model and the drug release from the tablets was controlled by degradation of the matrix. The preparation of hydrophilic matrix sustained release tablets of total lactones from A. paniculata with good performance of drug release was simple.

[Effects of fenofibrate on the growth and migration of ovarian cancer cells in vitro].

OBJECTIVE: To investigate the effects of fenofibrate, a lipid-lowering drug, on the growth and migration of human ovarian cancer cells SKOV3 in vitro. METHODS: A human ovarian cancer cell line (SKOV3) as the research object, was incubated with serum-free media for 24 h. These cells were then treated by appropriate concentrations of fenofibrate for different time, including control and experimental groups. Cell proliferation was evaluated by MTT assay. Apoptosis was detected by Hoechst/PI and Annexin-V/PI fluorescent assay. The migration of cells was measured by the scratch-wound healing assay. RESULTS: The MTT assay results demonstrated that the fenofibrate (10, 25, 50, 75, 100 micromol/L) could inhibit the proliferation of SKOV3 cells after 24, 48 and 72 h treatment (P < 0.05). The inhibition rate for 24, 48, 72 h-treatment was 55.72% +/- 0.28%, 57.63% +/- 0.47%, 72.41% +/- 0.62% respectively (P < 0.05). The effects increased with the concentrations. Hoechst/PI and Annexin-V/PI fluorescent assay showed that after stimulus for 24 h, fenofibrate induced apoptosis of SKOV3 cells in a concentration-dependent manner was observed. A significant inhibited cells migration distance (P < 0.05) evaluated with scratch-wound healing assay was observed after treatment with fenofibrate (10, 25, 50, 75, 100 micromol/L) for 24 h. CONCLUSION: Lipid-lowering drug fenofibrate can inhibit the growth and migration of human ovarian cancer cell SKOV3 in vitro, to some extent induce apoptosis. But the detailed mechanism need to be further studied.

Piperine suppresses tumor growth and metastasis in vitro and in vivo in a 4T1 murine breast cancer model.

AIM: To investigate the effects of piperine, a major pungent alkaloid present in Piper nigrum and Piper longum, on the tumor growth and metastasis of mouse 4T1 mammary carcinoma in vitro and in vivo, and elucidate the underlying mechanisms. METHODS: Growth of 4T1 cells was assessed using MTT assay. Apoptosis and cell cycle of 4T1 cells were evaluated with flow cytometry, and the related proteins were examined using Western blotting. Real-time quantitative PCR was applied to detect the expression of matrix metalloproteinases (MMPs). A highly malignant, spontaneously metastasizing 4T1 mouse mammary carcinoma model was used to evaluate the in vivo antitumor activity. Piperine was injected into tumors every 3 d for 3 times. RESULTS: Piperine (35-280 µmol/L) inhibited the growth of 4T1 cells in time- and dose-dependent manners (the IC(50) values were 105 ± 1.08 and 78.52 ± 1.06 µmol/L, respectively, at 48 and 72 h). Treatment of 4T1 cells with piperine (70-280 µmol/L) dose-dependently induced apoptosis of 4T1 cells, accompanying activation of caspase 3. The cells treated with piperine (140 and 280 µmol/L) significantly increased the percentage of cells in G(2)/M phase with a reduction in the expression of cyclin B1. Piperine (140 and 280 µmol/L) significantly decreased the expression of MMP-9 and MMP-13, and inhibited 4T1 cell migration in vitro. Injection of piperine (2.5 and 5 mg/kg) dose-dependently suppressed the primary 4T1 tumor growth and injection of piperine (5 mg/kg) significantly inhibited the lung metastasis. CONCLUSION: These results demonstrated that piperine is an effective antitumor compound in vitro and in vivo, and has the potential to be developed as a new anticancer drug.

Relationship between overexpression of NK-1R, NK-2R and intestinal mucosal damage in acute necrotizing pancreatitis.

AIM: To study the expression of neurokinin-1 receptor (NK-1R) and neurokinin-2 receptor (NK-2R) in distal ileum of acute necrotizing pancreatitis (ANP) and to evaluate the relationship between expression of these two receptors and intestinal mucosal damage. METHODS: A total of 130 adult Sprague-Dawley rats were randomly divided into two groups: the rats in ANP group (n=80) were induced by the retrograde intraductal infusion of 30 g.L(-1) sodium taurocholate. And the rats in normal control group (n=50) received laparotomy only. Sacrifices were made 6 h, 12 h, 24 h and 48 h later in ANP and normal control group after induction respectively. Intestinal mucosal permeability was studied by intrajejunal injection of 1.5 mCi radioactive isotope (99m)Tc-diethlene triamine pentacetic acid (DTPA) and the radioactivity of (99m)Tc-DTPA content in urine was measured 6 h, 12 h, 24 h and 48 h after induction. Then the pancreas and intestine were prepared for pathology. Reverse transcription polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of NK-1R and NK-2R, and Western blot was used to investigate the protein level of NK-1R and NK-2R. RESULTS: In ANP rats, serious histologic damages in intestinal mucosa were observed, and the radioactivity of (99m)Tc-DTPA in urine increased significantly in the ANP group. RT-PCR revealed that NK-1R and NK-2R mRNA level was overexpressed in the distal ileum of ANP as compared with the normal control group. Western blot discovered stronger NK-1R (14-fold increase) and NK-2R (9-fold increase) immunoreactivity in the intestinal mucosa of ANP rats. Moreover, the overexpression of NK-1R was associated with mucosal pathological score (r=0.77, P<0.01) and intestinal permeability (r=0.68, P<0.01) in ANP rats. CONCLUSION: NK-1R and NK-2R contribute to disrupted neuropeptides loop balance, deteriorate intestinal damage, and are involved in pathophysiological changes in ANP.

Clinical and experimental study on regional administration of phosphorus 32 glass microspheres in treating hepatic carcinoma.

AIM:To study the therapeutical effectiveness, dosage range and toxic adverse effects of domestic phosphorus 32 glass microsphere and evaluate its clinical significance.METHODS:I.Fifty two BALB/c tumor bearing male nude mice were allocated into treatment group(n = 38) and control group(n = 14). In the former group different doses of (32) P-GMS were injected into the tumor mass, while in the latter (31)P-GMS or no treatment was given. The experimental animals were sacrificed in batches, and then the tumors and their nearby tissues were examined by light and electron microscopy.II. Through selective catheterization of hepatic artery, (32)P-GMS was infused to 5 healthy domestic pigs in a dosage equivalent to the therapeutic dose for human being, and (31)P-GMS was infused to another 5 healthy domestic pigs. Two pigs infused with contrast medium served as whole course blank controls. One pig from each group was surrendered to euthanasia at week 1, 4, 8 and 16 respectively. The ultrastructural histopath ological changes in liver tissues taken from different sites were evaluated semiquan titatively. III. One hundred and twenty seven times of (32)P-GMS intrahepatic artery interventional therapies were performed on 93 patients with hepatic carcinoma, including 79 cases of primary hepatic carcinoma and 14 cases of secondary hepatic carcinoma. (32)P-GMS (n = 30), and group B,(32)P-GMS and half dose of trans hepatic artery embolization (TAE)(n = 49), and 18 patients with HCC by TAE only as control group C. Fourteen patients with secondary hepatic carcinoma were treated in the same way as group B or C.RESULTS:I.Comparing with the control group, the treatment group of tumor bearing nude mice attained the tumor inhibition rates of 59.7%-93.7% (F = 579.62 P < 0.01) at 14d. At an absorbed dose of 7320Gy, the tumor cells were completely destroyed. When the absorbed doses ranged from 1830Gy to 3660Gy, most of the tumor cells showed the evidences of injury or necrosis, but there appeared some well differentiated tumor cells and enhanced effect of the autoimmunocytes. At an absorbed dose of 366Gy or less, some tumor cells still remained active prolix-ferative ability. The definite anticancer effect appeared as early as 3d after intratumoral injection of (32)P-GMS.II. The cumulative amount of (32) P-GMS in the target tissue after trans hepatic artery instillation attained more than 90% of the total dose administrated. Semiquantitative analysis of ultrastructral morphology in the experimental group showed no statistical difference between the nuclear abnormality (N(abn)) and mitochondrial variability (M(var)) at week 1 or 2, but revealed prominent difference (X(2) = 6.70-9.68, P < 0.01, X(2) = 65.09-115.09, P <0.001) as compared with those in the other groups. In the experimental group the N abn in tissues showed no significant difference between week 8 and week 16. No apparent changes were found in the stomach, spleen, kidney and lung tissues of the experimental pigs. III. The therapeutical results of HCC patients in group A were closely approximated to those of group C, no hematological toxic side effects were noted, and the systemic reaction was mild. In some patients 2mos-3mos after treatment some secondary foci appeared around the periphery of the primary lesion. In general better effectiveness was obtained in patients with small lesion. After analyzing by RIDIT method, the therapeutic result in group B was significantly better than that in group C, and secondary foci around the original lesion were rarely seen at 3mos after treatment. In group C the collateral circulation was reestablished along the periphery of primary foci and the secondary foci appeared more frequently, and were required to undergo several courses of treatment. In group B, 4 cases of HCC were treated surgically as their mass decreased in size after (32)P-GMS treatment.Resected specimens showed that the tumor was encapsulated by fibrotic tissue and most of the tumor cells necrosed. The 3 year survival rates were 43.3%-51.0% after A and B regimen treatment. In 14 cases of secondary HCC, the foci were well controled within one year after treatment.CONCLUSION:When the experimental model of implanted human liver cancer cells received (32)P-GMS of 1830Gy-3660Gy, it produced excellent anticancer effect without any injury to the normal neighboring tissues and the prominent anticancer effect was shown within 3d after intratumoral injection. Intrahepatic arterial administration of (32)P-GMS at the macro-cosmic absorbed dosage less than 190 Gy/dose exerted reversible sub lethal injury to domestic pig liver tissues. It took more than 8 weeks to repair the injured liver tissue and restore its function.(32)P-GMS trans hepatic artery embolization is an effective and safe regimen in treating hepatic carcinoma.