Enhanced antitumor efficiency of docetaxel-loaded nanoparticles in a human ovarian xenograft model with lower systemic toxicities by intratumoral delivery

BackgroundThe aim of the study was to investigate the suppressive effects of pSilencer2.1-U6-siRNA-stat3 recombinant plasmids on the growth of ovarian cancer in vitro.Material and methods.Three pairs of DNA template (stat3-1, stat3-2, stat3-3) specific for different target sites on stat3 mRNA were synthesized to reconstruct pSilencer2.1-U6-siRNA-stat3s, which were transfected into SKOV3 cells. The expressions of STAT3, BcL-2, cyclin D1 and C-myc in these cells were detected by Western blot and Northern blot. The cell cycle and the growth were determined by flow cytometry (FCM) and MTT assay, respectively. Cell apoptosis was determined by TUNEL staining.ResultsOf the three siRNAs, only siRNA targeting stat3-3 markedly suppressed the protein expression of stat3 in SKOV3 cells; MTT assay and FCM showed that transfection of stat3-3 siRNA could significantly suppress the growth of SKOV3 cells and arrest the cell cycle in vitro. TUNEL staining also showed massive apoptosis in SKOV3 cells transfected with stat3-3 siRNA.ConclusionspSilencer2.1-U6-siRNA-stat3-3 can significantly inhibit the STAT3 expression in human ovarian cancer cells resulting in the inhibition of the cancer growth and the increase of apoptosis of cancer cells.

Superior antitumor efficiency of cisplatin-loaded nanoparticles by intratumoral delivery with decreased tumor metabolism rate

Inhibition of growth of human ovarian cancer in nude mice by luteinizing hormone-releasing hormone antagonist Cetrorelix (SB-75 )

We have recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) ligands produce antitumor effects against human ovarian cancer in conjunction with reduction in angiogenesis and induction of apoptosis via regulating prostaglandin (PG) E(2) level. In this study, we investigated the effects of combination of ciglitazone, a PPARγ ligand, and cisplatin, a cytotoxic anti-cancer drug, on growth of ovarian cancer. Tumor growth and survival were examined in female nu/nu mice xenografted with subcutaneous OVCAR-3 tumors or with intraperitoneal DISS tumors and treated with cisplatin alone (5mg/kg intraperitoneally once on day 1), ciglitazone alone (15mg/kg intraperitoneally once a week), or the combination. Ciglitazone alone, cisplatin alone, or their combination significantly suppressed the growth of OVCAR-3 tumors xenotransplated subcutaneously and prolonged the survival of mice with malignant ascites derived from DISS cells as compared with the control. Furthermore, the combination produced a significantly greater antitumor effect than cisplatin or ciglitazone alone and also significantly prolonged the survival time as compared with cisplatin or ciglitazone alone. The combination significantly decreased PGE(2) concentration in serum as well as in ascites, reduced vascular endothelial growth factor as well as microvessel density, and induced apoptosis in solid OVCAR-3 tumor as compared with cisplatin or ciglitazone alone. The combination remarkably decreased the expression of cyclooxygenase-2 (COX-2), microsomal PG E synthase (mPGES), and PG receptor 3 (EP3) in tumors. In vitro experiment showed that ciglitazone enhances the cytotoxicity of cisplatin against ovarian cancer cells. In conclusion, the combination inhibited the growth of ovarian cancer in conjunction with reduction in angiogenesis and induction of apoptosis resulting from suppression of PGE(2) activation through decreasing the expression of COX-2, mPGES, and EP3. The inhibitory effect of this combination treatment on growth of ovarian cancer suggests a potential to lead a novel therapeutic strategy against ovarian cancer.

Successful chemotherapy needs to reduce the toxic side effects against normal tissues and avoid the detriments caused by intolerable solvents. Drug delivery systems using soluble polymeric nanoparticles tend to be the focus. In the current study, core-shell structure nanoparticles were prepared from block copolymer of methoxy poly(ethylene glycol)-polycaprolactone (mPE-PCL). Paclitaxel (PTX) and berbamine (BA) were incorporated into mPEG-PCL nanoparticles. It was found in our study that PTX and BA can be incorporated into the nanoparticles with high encapsulation efficiency. In vitro release study showed that PTX and BA were released from nanoparticles in a sustained manner. In vitro cytotoxicity studies indicated that PTX/BA coloaded nanoparticles (PTX/BA-np) show dose- and time-dependent cytotoxicity again BGC823 cells. Furthermore, intratumoral administration was applied to improve the tumor-targeted delivery in the in vivo evaluation. Compared with free drugs, PTX/BA-np exhibited superior antitumor effect by delaying tumor growth when delivered intratumorally. These results suggest that PTX/BA-np are effective to inhibit the growth of human gastric cancer and merit more research to evaluate the feasibility of clinical application.

This study investigated the inhibitory effects of miR-124 and miR-152 on the growth of human ovarian cancer (OC) SKOV3 cell line subcutaneous xenografts in nude mice. Twenty-eight healthy nude mice were selected and divided into the experimental group 1 (n=4), experimental group 2 (n=4), negative control group 1 (n=4), negative control group 2 (n=4), blank control group 1 (n=4), blank control group 2 (n=4) and observation group (n=4) according to the principle of similarity in body weight. The transfected SKOV3 cells were inoculated subcutaneously into the nape of the nude mice. After tumorigenesis, miR-124 mimics, miR-152 mimics, and their negative controls were transiently transfected into human OC SKOV3 cells via lipofection method. The expression levels of miR-124 and miR-152 were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and those of Ki-67 and caspase-3 were detected by western blotting. After transfection, the expression levels of miR-124 and miR-152 in the SKOV3 cells were significantly upregulated. The nude mice were sacrificed 36 days later, and tumor nodes of nude mice transfected with miR-124 and miR-152 grew slowly. Compared with that in the experimental groups, tumor size in the blank control and negative control groups was gradually increased with the increment of days (P<0.05). The volume of subcutaneous xenografts in nude mice of miR-124 and miR-152 experimental groups was obviously smaller than that in the blank control and negative control groups (P<0.05). Besides, the inhibition of tumor size in the observation group was more significant than that in the experimental groups (P<0.05). Thus, miR-124 and miR-152 inhibit the growth of human epithelial OC xenografts in nude mice, and they are expected to become new targets for gene-based therapy of OC.

Tumor growth and metastasis involves complex crosstalk among cancer cells and immune infiltrates. The balance of pro- and antitumor infiltrating immune cells can impact the outcomes of tumor growth. Vacuolar-ATPase (V-ATPase) proton pump promotes cancer growth and metastasis by regulating the pH-associated cancer cell-signaling and the tumor microenvironment. V-ATPase is overexpressed on cancer cell surface in a wide array of tumor types including ovarian cancer (OVCA) as well as on inflammatory cells recruited to the tumor microenvironment. This makes V-ATPase an important target for anticancer therapy. We have created a monoclonal antibody that specifically recognizes the plasma membrane isoform of V-ATPase (a2v) present distinctly on malignant cells and absent on normal cells. The purpose of this study is to investigate the in vivo effect of anti-VATPase-a2v antibody (Mab; 2C1) on the growth of human ovarian cancer (OVCA). The antitumor effects were evaluated in female athymic nude mice xenograft model. Mice were injected with human OVCA cell line (A2780;0.4X106 cells; s.c in upper flank) and the tumor growth was compared to mice that were simultaneously given a single dose of MAb (2C1; 300µg in PBS) or an isotype control antibody(mouse IgG1A). The 2C1 treatment resulted in delayed tumor growth (2.4 fold, p&amp;lt; 0.05), with no apparent in vivo toxicity. Histologic staining of 2C1-treated tumors revealed a loose tumor matrix with higher immune-infiltration compared to control. There was a marked decrease in cancer cell numbers in 2C1-treated tumor tissues compared to control using CA125 cancer-antigen staining. In in vitro assays, there was no measurable cytotoxic effect of 2C1 on OVCA cell proliferation, indicating the role of tumor microenvironment in delayed tumor growth. We further investigated the infiltrated immune population in 2C1-treated tumors. Interestingly, we observed an overall increased total leukocyte population (CD45; p=0.028) and a higher expression of iNOS (IHC-score 2C1 treated tumor= 11.3, control =7.6; p=0.04), suggesting an antitumor response. The total F4/80 macrophage population was higher in anti-VATPase-a2v antibody treated cells (absolute numbers in 2C1 treated tumor=245.2, control tumor=193.1; p=0.04). In 2C1 treated mice, tumor-associated macrophages displayed antitumor properties with correlated iNOS expression. Further, a marked increase in the number of Ly6G positive cells (neutrophil marker) was observed in 2C1 treated tumors, further suggesting an elevated antitumor response. Studies are under way to characterize other immune infiltrated populations that may contribute to antitumor response in 2C1 treated tumor tissues. In conclusion, the study demonstrates that the anti-VATPase "a2" antibody is an effective form of treatment in ovarian cancer. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Shayna Levine, Manoranjan Sahoo, Safaa A. Ibrahim, Alice Gilman-Sachs, Kenneth D. Beaman. Cancer growth under check: Anti-vacuolar ATPase "a2" antibody as novel therapy against ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2913.

Epithelial ovarian carcinoma is the leading cause of cancer-related deaths among women with gynecologic malignancies. Antagonists of the growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of various cancers through endocrine, autocrine, and paracrine mechanisms. In this study, we have investigated the effects of GHRH antagonists (GHRHa) in ES-2 human clear cell ovarian cancer and in UCI-107 human serous ovarian cancer in vitro and in vivo. We evaluated the expression of mRNA for GHRH receptor, the binding to GHRH receptors, in specimens of ES-2 ovarian cancer. We evaluated also the in vitro effects of GHRHa on ES-2 cells and the in vivo effect of 2 different GHRHa on ES-2 and UCI-107 tumors. Nude mice bearing xenografts on ES-2 and UCI-107 ovarian cancer were treated with JMR-132 and MZ-J-7-118, respectively. Tumor growth was compared to control. ES-2 cells expressed mRNA for the functional splice variant SV1 of the GHRH receptor. JMR-132 inhibited cell proliferation in vitro by 42% and 18% at 10 and 1 μM concentration, respectively. Specific high affinity receptors for GHRH were detected in ES-2 cancer samples. In vivo daily subcutaneous injections of GHRHa significantly reduced tumor growth compared to a control group in both animal models. Our results indicate that GHRHa such as JMR-132 and MZ-J-7-118 can inhibit the growth of human ovarian cancer. The efficacy of GHRHa in ovarian cancer should be assessed in clinical trials.

Decreased concentrations of pigment epithelium–derived factor in peritoneal fluid of patients with endometriosis

Introduction: We aimed to explore small interfering (si)RNA silencing of ribonucleotide reductase M2 (RRM2) gene combined with cisplatin for the treatment of human ovarian cancer in nude mice models of subcutaneous transplantation of tumor cells.Methods: After conventional cultivation of human ovarian cancer cell line SKOV3 in vitro, SKOV3 cells were injected into the right back of nude mice by subcutaneous injection to establish the subcutaneous tumor models. Twenty-four tumor-burdened rats were randomly divided into four groups (n=6): siRNA group, siRNA in combination with cisplatin group, cisplatin group, and control group. Intraperitoneal injection of cisplatin and subcutaneous injection of siRNA were performed weekly. Tumor volume was measured, and tumor growth inhibition rate was calculated. RRM2 expression at the mRNA and protein levels was detected by reverse transcription-polymerase chain reaction and immunohistochemistry.Results: In the siRNA group, the tumor volume and tumor growth inhibition rate were 249.60±20.46 mm³ and 36.39%, respectively. The tumor growth inhibition rate and tumor volume were significantly different between the siRNA and control groups (p<0.05). In the cisplatin group, the tumor volume and tumor growth inhibition rate were 249.86±12.46 mm³ and 41.10%, respectively. The tumor growth inhibition rate and tumor volume were significantly different between the cisplatin and control groups (p<0.05). In the siRNA + cisplatin group, the tumor volume reduced to 180.84±16.25 mm³ and the tumor growth inhibition rate was increased to 64.33%, which were significantly different compared with the control group (p<0.01). Significant downregulation of RRM2 mRNA and protein expression in the tumor tissues was detected by reverse transcription polymerase chain reaction and immunohistochemistry assay (p<0.05).Discussion: siRNA alone or combined with cisplatin can effectively inhibit the growth of human ovarian cancer in nude mice models of subcutaneous transplantation of tumor cells. RRM2 gene silencing may be a potential treatment regimen for ovarian cancer in future.

To evaluate the potential effect of anticancer and antiangiogenesis of Stx1(W203F) and Stx1(R170H), two attenuated mutants of Shiga-like toxin I (Stx1), in cancer gene therapy. Antiproliferative effects of these Stx1 mutants were tested in human ovarian carcinoma cell line SKOV3 and human umbilical vein endothelial cells (HUVECs) in vitro. Effect of these Stx1 mutants on inducing cell death and cell cycle arrest was analyzed in SKOV3 cells. Influence of these Stx1 mutants on endothelial cell function was analyzed in HUVECs. In vivo therapeutic effect of these Stx1 mutants on SKOV3 was explored using xenograft models in nude mice. These Stx1 mutants can inhibit the growth of SKOV3 or HUVECs and this effect can be abrogated by antibody specific for Stx1. They caused considerable cell death of SKOV3 cells in 24 h; neither caspase activity nor DNA fragmentation was observed, and necrosis is the major mode of cell death. These Stx1 mutants can induce cell cycle arrest of SKOV3 cells in G(2)-M or S phase depending on the dosage of gene transfer. Furthermore, they significantly decreased migration and capillary tube formation of HUVECs at low dose. In vivo study showed that Stx1(W203F) but not Stx1(R170H) significantly suppressed transplanted SKOV3 tumor growth in nude mice model. Interestingly, the microvessel densities of tumor treated with Stx1(W203F) and Stx1(R170H) were significantly reduced. This study suggests that genes encoding attenuated Stx1 can be selected as good candidates for the gene therapy of ovarian carcinoma because of their antiproliferative and antiangiogenic effects.

Efficient antiproliferative and antiangiogenic effects on human ovarian cancer growth by gene transfer of attenuated mutants of Shiga-like toxin I

It was recently reported that high expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and low expression of cyclooxygenase-2 (COX-2) might be involved in the inhibition of ovarian tumor progression and confirmed that PPARgamma activation could suppress COX-2 expression via the nuclear factor-kappaB pathway in ovarian cancer cells. The current study investigated whether meloxicam, a selective COX-2 inhibitor, and ciglitazone, a ligand for PPARgamma, inhibit the growth of human ovarian cancer cell lines and aimed to elucidate the molecular mechanism of their antitumor effect. Tumor growth and survival were examined in female nu/nu mice xenografted with subcutaneous OVCAR-3 tumors or with intraperitoneal DISS tumors and treated with meloxicam (162 ppm in diet, every day) or ciglitazone (15 mg/kg intraperitoneally once a week). Both meloxicam and ciglitazone treatments significantly suppressed the growth of OVCAR-3 tumors xenotransplanted subcutaneously and significantly prolonged the survival of mice with malignant ascites derived from DISS cells as compared with controls. Meloxicam treatment decreased COX-2 expression in tumors by 2.5-fold compared with that observed in untreated tumors. Although ciglitazone treatment did not alter COX-2 expression in tumors, it reduced the expression of microsomal prostaglandin (PG) E synthase, which converts COX-derived PGH(2) to PGE(2). Both meloxicam and ciglitazone decreased PGE(2) levels in serum as well as in ascites. Reduced microvessel density and induced apoptosis were found in solid OVCAR-3 tumors treated with either meloxicam or ciglitazone. These results indicate that both meloxicam and ciglitazone produce antitumor effects against ovarian cancer in conjunction with reduced angiogenesis and induction of apoptosis.

ObjectiveThe aim of this study was to determine the effects and possible mechanisms of oldhamianoside on the growth of human ovarian cancer both in vitro and in vivo.Materials and methodsCCK-8 assay was applied to estimate the effect of oldhamianoside on cell proliferation inhibition in vitro. Nude mice bearing human ovarian SKOV3 xenograft tumors were treated with oldhamianoside to investigate the effects of compound administration on tumor growth in vivo. To further investigate the mechanisms of inhibition effects of oldhamianoside on ovarian cancer growth in vivo, the levels of TNF-α, IL-6, and MCP-1 in plasma from the mice were measured by ELISA. Western blot was used to detect the expression of angiogenesis- and/or apoptosis-related proteins.ResultsWe found that oldhamianoside treatment inhibited SKOV3 proliferation and growth both in vitro and in vivo. Meanwhile, the levels of TNF-α, IL-6, and MCP-1 in plasma were markedly suppressed in oldhamianoside-treated mice. Additionally, oldhamianoside treatment inhibited the expression of VEGF and VEGFR2 and decreased the expression of caspase-3 and Bax/Bcl-2 ratio.ConclusionOur data indicate that oldhamianoside has an obvious inhibition effect on SKOV3 proliferation, and the mechanisms might be related to inhibition of cell growth, apoptosis induction, and adjusting the inflammatory response and angiogenesis signal.

In a previous study, we screened organic extracts of different mushroom mycelia and picked ethyl acetate extract of Coprinus comatus as one of the most active extracts against human ovarian cancer cells. In the current study, we extracted a dry powder of C. comatus fruit bodies using ethyl acetate and examined its effect on the viability of three cell lines originated from human ovarian cancer (ES-2, SKOV-3, and SW-626). This extract was active against all tested cell lines, in a dose-dependent manner (concentrations 50-200 µg/mL, P<0.01). In an attempt to segregate the active fraction, we subjected the extract to chromatography on a silica gel column. The effect of six different fractions and of the crude extract on the viability of ES-2 cells was examined after exposure time of 24 h. Fraction F (last eluted) was significantly more effective than crude extract in the reduction of cell viability (P<0.01). Fraction F was also significantly more active than crude extract in the reduction of viability of SKOV-3 cells. We next identified some of the compounds of fraction F (mainly fatty acids) by gas chromatography-mass spectrometry. In summary, ethyl acetate extract of C. comatus reduced viability of three lines of human ovarian cancer. Fractionation of this extract by a silica gel column enabled the selection of a fraction significantly more active than the original extract.