Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites: An Experimental Study.

OBJECTIVE: This study aimed to explore the value of M701, targeting epithelial cell adhesion molecule (EpCAM) and CD3, in the immunotherapy of ovarian cancer ascites by the in vitro assay. METHODS: The expression of EpCAM in ovarian cancer tissues was analyzed by databases. The EpCAM expression and immune cell infiltration in different foci of ovarian cancer were detected by 8-channel flow cytometry. The toxic effect of M701 on OVCAR3 was tested using the in vitro cytotoxicity assay. The 3D cell culture and drug intervention experiments were performed to evaluate the therapeutic effect of M701 in ovarian cancer specimens. Flow cytometry was used to examine the effect of M701 on the binding of immune cells to tumor cells and the activation capacity of T cells. RESULTS: The results of the bioinformatic analysis showed that the expression of EpCAM in ovarian cancer tissue was significantly higher than that in normal ovarian tissue. The 8-channel flow cytometry of clinical samples showed that the EpCAM expression and lymphocyte infiltration were significantly heterogeneous among ovarian cancer patients and lesions at different sites. The in vitro experiment results showed that M701 had a significant killing effect on OVCAR3 cells. M701 also obviously killed primary tumor cells derived from some patients with ovarian cancer ascites. M701 could mediate the binding of CD3+ T cells to EpCAM+ tumor cells and induce T cell activation in a dose-dependent manner. CONCLUSION: M701 showed significant inhibitory activity on tumor cells derived from ovarian cancer ascites, which had a promising application in immunotherapy for patients with ovarian cancer ascites.

[Antitumor and antimetastatic effect of antimicrobial peptide conjugated with tumor homing peptide TMTP1 on the transplanted prostate cancer and gastric cancer in nude mice].

OBJECTIVE: Due to their lower risk for induction of resistance, antimicrobial peptides with selective anticancer effect could be developed into a new generation of anticancer drugs. We conjugated an antimicrobial peptide with tumor-targeting peptides (TMTP1) to explore whether it has inhibiting effect on the progression and metastasis of transplanted prostate cancer and gastric cancer in nude mice. METHODS: Subcutaneously transplanted human prostate cancer and orthotopically transplanted human gastric cancer in nude mice were prepared. 50 µmol/L PBS (control group), 50 µmol/L TMTP1 (TMTP1 group) or 50 µmol/L TMTP1-GG-D(KLAKLAK)(2) (treatment group) were injected i.p. to the three groups of nude mice, respectively. The binding ability of the novel fusion polypeptide TMTP1-GG-D(KLAKLAK)(2) to the tumors and its antitumor effect were assessed by measurement of tumor volume, histopathological examination of the tumor tissues, testing apoptosis index of tumor cells with TUNEL staining, and survival curve plotting of the mice. RESULTS: The median survival time of subcutaneous prostate cancer-bearing mice was 50 days in the control group, 55 days in the TMTP1 group, and 70 days in the TMTP1-GG-D(KLAKLAK)(2) group (P < 0.05). The median survival time of the subcutaneous gastric cancer-bearing mice was 25 days in the control group, 30 days in the TMTP1 group, and 45 days in the TMTP1-GG-D(KLAKLAK)(2) group (P < 0.01). The tumor volume in the subcutaneous prostate cancer-bearing mice was (2.5 ± 0.3)cm(3) in the control group, (1.8 ± 0.2) cm(3) in the TMTP1 group, and (0.3 ± 0.1)cm(3) in the TMTP1-GG-D(KLAKLAK)(2) group (P < 0.01). The tumor volume of the subcutaneous gastric cancer-bearing mice was (3.8 ± 0.4) cm(3) in the control group, (3.2 ± 0.2)cm(3) in the TMTP1 group, and (0.4 ± 0.1) cm(3) in the TMTP1-GG-D(KLAKLAK)(2) group (P < 0.01). Large tumors were observed in the stomach of the orthotopic gastric cancer-bearing mice of the control and TMTP1 groups. The tumor volume of the TMTP1-GG-D(KLAKLAK)(2) group was obviously reduced. White metastases in the liver, spleen and abdominal wall were observed in the control and TMTP1 groups (P < 0.01). TUNEL staining revealed that the apoptosis index of the control group was (31.9 ± 1.5)%, TMTP1 group (37.2 ± 2.3)% and TMTP1-GG-D(KLAKLAK)(2) group (69.7 ± 2.1)% (P < 0.01). CONCLUSIONS: The results of our study demonstrate that the novel fusion peptide of antimicriobial peptide conjugated with TMTP1 can effectively inhibit tumor progression and metastasis, therefore, is promising to be a novel effective anticancer drug.

[The role of posterolateral structure and posterior cruciate ligament in external rotatory stability of the knee].

OBJECTIVE: To investigate the changes of external rotation stability of the knee after the breaking and reconstruction of the posterolateral structure (PLS) of knee joint. METHODS: The femurs of 16 fresh cadaveric lower limbs were fixed to the base of rotating holders with the knee joints in full extension, or at an angle of 30 degrees , 60 degrees , or 90 degrees ; while the tibia was attached to a free rotary holder. The external rotation of the tibia was measured with a 5 kg x m x s(-2) tibial torque. The external rotation angle of the tibia was measured after the transectioning of the popliteus tendon (PT), popliteus muscle (PM), popliteofibular ligament (PFL), fibular collateral ligament (LCL), or popliteus tendon (PT). Autogenous hamstring tendon was used to reconstruct the PCL, and the Achilles tendon was used to reconstruct the PFL and PT, then the external rotation angle of the tibia was measured again. RESULTS: The isolated sectioning of PCL did not increase the tibial external rotation angle. When the knee was flexed the external rotation angle was 14.1 degrees in the intact knee and was 14.57 degrees in the PCL transectioning group (q = 0.47, P > 0.05). After the PFL was sectioned, when the knee joint was flexed at 60 degrees the external rotation angle was 16.94 degrees (q = 2.84, P < 0.05). After the PT was sectioned (PCL + PFL + PT), when the knee joint was flexed at 60 degrees the external rotation angle was 28.1 degrees (q = 14.01, P < 0.05). After isolated PCL reconstruction the external rotation angle was still bigger than that of the normal knee: for example, when the knee joint was at the angle of 60 degrees the external rotation angle was 27.67 degrees (q = 0.425, P < 0.05). There was a significant decrease in external rotation compared with PT section (P < 0.05). After the combined PCL + PLS reconstruction when the knee joint was at the angle of 60 degrees the external rotation angle of the knee was 14.51 degrees (q = 0.412, P < 0.05), however, the range of change was less than 1 degree. CONCLUSION: Isolated PCL section produces no change in external rotation. Complex injury of PCL and PSL can produce instability in external rotation. PT has the greatest important role to resist external rotation, then the greatest increase in external rotation can be found after PT is sectioned. Isolated PCL reconstruction can not completely restore the posterolateral stability of the knee. PCL reconstruction can partly restore the stability of the external tibial rotation. Only the combined PC + PLS reconstruction can reset the knee to physiological stability of external rotation.

[Induction effect of rapamycin combined paclitaxel on apoptosis of ovarian cancer cell lines A2780 and SKOV3 and the molecular mechanism].

BACKGROUND & OBJECTIVE: Previous researches confirmed that the mammalian target of rapamycin (mTOR) plays an important role in the tumorigenesis and development of malignant tumors. This study was to investigate the effect of rapamycin, a selective inhibitor of mTOR, combined paclitaxel on the apoptosis of ovarian cancer cell lines A2780 and SKOV3, and explore the molecular mechanism. METHODS: A2780 and SKOV3 cells were treated with rapamycin and (or) paclitaxel. Cell proliferation was assessed by MTT assay. The interaction of rapamycin and paclitaxel was estimated by Jin Zhengjun's method. Cell apoptosis was detected by flow cytometry (FCM). The expression of survivin in A2780 and SKOV3 cells was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: When treated with rapamycin combined paclitaxel for 72 h, the proliferation inhibition rate was 34.9% for A2780 cells and 37.1% for SKOV3 cells, which was significantly higher than those of the cells treated with rapamycin or paclitaxel alone (P<0.01). These 2 drugs showed synergistic effect (q>1.15). The apoptosis of A2780 and SKOV3 cells were induced by rapamycin and paclitaxel; the apoptosis rate reached to the peak when the cells were treated with rapamycin combined paclitaxel. The expression of survivin in A2780 and SKOV3 cells was declined obviously after treatment of rapamycin combined paclitaxel. CONCLUSIONS: Rapamycin and paclitaxel could inhibit proliferation and induce apoptosis of A2780 and SKOV3 cells in vitro, and down-regulate the expression of survivin. These 2 drugs have synergistic effect on cell proliferation.

[Effects of selected phages with specific peptide ligands on the biological behaviors of ovarian cancer cells].

OBJECTIVE: To evaluate the effects of the selected phages with the specific peptide ligands upon the biological behaviors of the ovarian cancer cells. METHODS: Two ovarian cancer cell lines, A2780 and SKOV3, were used in the study. They were divided into three groups respectively: study group, blank control group and negative control group. The effects of the phages were evaluated by trypan blue staining, flow cytometry, colony formation test, methyl thiazolyl tetrazolium (MTT) assay and Boyden chamber invasion assay. RESULTS: The average viability of ovarian cancer cells of study groups was (72.1 +/- 6.2)%, higher than that of negative control group (84.8 +/- 4.6, P < 0.05); the apoptosis ratio of the A2780 and SKOV3 cells of study groups increased (20.39% and 43.99% vs 0); the average colony formation rate of the ovarian cancer cells was of study groups 4%, lower than the control group (15%, P < 0.05); the inhibition rates of cell proliferation of A2780 and SKOV3 cells of study groups were 11.07% and 9.58%, significantly higher than that of their respective negative control (2.05% and 1.09%); the invasion index of the ovarian cancer cells decreased compared with the control. CONCLUSION: The selected phages down-regulate the growth, proliferation and invasion ability of the ovarian cancer cells to certain extent, the identified peptide ligands may play a role in tumorigenesis and metastatic transformation of ovarian cancers.

[Expression of survivin in ovarian epithelial carcinoma and its correlation with expression of Fas and FasL].

BACKGROUND & OBJECTIVE: Recent research had shown that survivin, a new member of the inhibitors of apoptosis proteins (IAP) family, which also plays an important role in mitosis and cell apoptosis, and selectively overexpressed in common human cancers. This study was designed to examine the expression of survivin and its correlation with expression of Fas and FasL in ovarian epithelial carcinoma. METHODS: Immunohistochemical assay (SP method) was used to detect the expression of survivin, Fas and FasL genes in 84 ovarian cancer tissues, 39 benign tumors of ovary, and 20 normal ovary tissues. RESULTS: The level of expression of survivin was higher in the patients with ovarian cancer (63.1%) than that in the patients with benign tumor of ovary (30.8%) and normal ovary tissues (0.0%)(P< 0.01). The expression level of survivin was strongly correlated with clinical stage and poor differentiation. The expression of Fas in ovarian cancer (23.8%) was significantly lower than that of ovarian benign tumor (53.8%) (P< 0.01). The expression rate of FasL was significantly higher in ovarian cancer (44.0%) than that in ovarian benign tumor (23.1%)(P< 0.05). Positive survivin expression was strongly correlated with Fas and FasL expression. CONCLUSION: (1) High expression of survivin may plays an important role in the development of ovarian cancer and could be a useful prognostic maker for patients with ovarian cancer.(2) The abnormal expression of Fas and FasL in ovary cancer and their correlation with survivin suggested that survivin might be in cooperation with Fas and FasL,which involved in the pathogenesis of ovarian cancer.

[Selection of targets locating on the surface of epithelial ovarian cancer cells by using phage peptide library].

OBJECTIVE: The aim of this study was to select specific targets locating on the surface of epithelial ovarian cancer cells. METHODS: Peptide phage display library was used to isolate specific ligand to ovarian cancer cell receptors. The diluted library was incubated with the normal ovarian cells that primarily cultured before hand, and then the supernatant of nonbonding phage was added to the first epithelial ovarian cancer cell line A2780. Phage that binds to the cell surface are eluted and then amplified to be used in the next round. After the third round of panning, the elute was used as input phage for the next cell line biopanning. Four epithelial ovarian cancer cell lines were used one by one in this way. Finally the positive clones were identified by ELISA assay. Sequencing analysis was carried out for further identification. RESULTS: 10 positive clones were chosen and one was regarded as target clone. A candidate sequence (YYGLAEVDAGGS) was identified by amino acid sequence assay. CONCLUSION: The phage peptide library provides an efficient selection system for searching special targets locating on the cell surface.