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DNA methylation plays a crucial role in the pathogenesis of various diseases, including colorectal cancer (CRC). However, the global and temporal DNA methylation pattern during initiation and progression of colitis-associated cancer (CAC) are still unknown, including the potential therapeutic strategy of targeting methylation for CAC. In the present study, the global DNA methylation pattern was determined at different time points during CAC using DNA methylation sequencing, followed by the Starburst plot integrating alterations and potential functional prediction analysis. After demonstrating the regulatory role of DNA methyltransferases (DNMTs) on the expression of hub-genes in CRC cells, DNMT inhibitors were administered to treat CAC mice. Our results indicated that 811 genes were hypermethylated at different time points during initiation and progression of CAC. Genes that were downregulated and hypermethylated during CAC, including hub-genes BAD and inositol polyphosphate phosphatase-like 1 (INPPL1), were involved in MAPK signaling pathways, kit receptor signaling pathways, apoptosis and EGF/EGFR signaling pathways. Upregulated DNMTs (DNMT1, DNMT3A and DNMT3B) mediated downregulation and hypermethylation of BAD and INPPL1 in CAC and CRC cells. Low doses of DNMT inhibitors (decitabine (DAC) and azacitidine (AZA)) exerted efficient antitumor effects in CAC, accompanied with upregulation of BAD and INPPL1 expression, and apoptosis induction. In summary, the present study demonstrates the temporal DNA methylation pattern during CAC and provides a novel therapeutic strategy for treating this disease.

SARI (suppressor of AP‐1, regulated by IFN) impaired tumour growth by promoting apoptosis and inhibiting cell proliferation and tumour angiogenesis in various cancers. However, the role of SARI in regulating tumour‐associated inflammation microenvironment is still elusive. In our study, the colitis‐dependent and ‐independent primary model were established in SARI deficiency mice and immuno‐reconstructive mice to investigate the functional role of SARI in regulating tumour‐associated inflammation microenvironment and primary colon cancer formation. The results have shown that SARI deficiency promotes colitis‐associated cancer (CAC) development only in the presence of colon inflammation. SARI inhibited tumour‐associated macrophages (TAM) infiltration in colon tissues, and SARI deficiency in bone marrow cells has no observed role in the promotion of intestinal tumorigenesis. Mechanism investigations indicated that SARI down‐regulates p‐STAT1 and STAT1 expression in colon cancer cells, following inhibition of MCP‐1/CCR2 axis activation during CAC development. Inverse correlations between SARI expression and macrophage infiltration, MCP‐1 expression and p‐STAT1 expression were also demonstrated in colon malignant tissues. Collectively, our results prove the inhibition role of SARI in colon cancer formation through regulating TAM infiltration.

Mesenchymal stem cell (MSC)-mediated immunomodulation has been harnessed for the treatment of human diseases, but its underlying mechanism has not been fully understood. Dead cells, including apoptotic cells have immunomodulatory properties. It has been repeatedly reported that the proportion of nonviable MSCs in a MSC therapeutic preparation varied from 5~50% in the ongoing clinical trials. It is conceivable that the nonviable cells in a MSC therapeutic preparation may play a role in the therapeutic effects of MSCs. We found that the MSC therapeutic preparation in the present study had about 5% dead MSCs (DMSCs), characterized by apoptotic cells. Namely, 1 × 106 MSCs in the preparation contained about 5 × 104 DMSCs. We found that the treatment with even 5 × 104 DMSCs alone had the equal therapeutic effects as with 1 × 106 MSCs. This protective effect of the dead MSCs alone was confirmed in four mouse models, including concanavalin A (ConA)- and carbon tetrachloride (CCl4)-induced acute liver injury, LPS-induced lung injury and spinal cord injury. We also found that the infused MSCs died by apoptosis in vivo. Furthermore, the therapeutic effect was attributed to the elevated level of phosphatidylserine (PS) upon the injection of MSCs or DMSCs. The direct administration of PS liposomes (PSLs) mimic apoptotic cell fragments also exerted the protective effects as MSCs and DMSCs. The Mer tyrosine kinase (MerTK) deficiency or the knockout of chemokine receptor C–C motif chemokine receptor 2 (CCR2) reversed these protective effects of MSCs or DMSCs. These results revealed that DMSCs alone in the therapeutic stem cell preparation or the apoptotic cells induced in vivo may exert the same immunomodulatory property as the “living MSCs preparation” through releasing PS, which was further recognized by MerTK and participated in modulating immune cells.

Transcription factors are known to mediate the conversion of somatic cells to induced pluripotent stem cells (iPSCs). Transcription factor TFAP2C plays important roles in the regulation of embryonic development and carcinogenesis; however, the roles of Tfap2c in regulating somatic cell reprogramming are not well understood. Here we demonstrate Tfap2c is induced during the generation of iPSCs from mouse fibroblasts and acts as a facilitator for iPSCs formation. Mechanistically, the c-Myc-dependent apoptosis, which is a roadblock to reprogramming, can be significantly mitigated by Tfap2c overexpression. Meanwhile, Tfap2c can greatly promote mesenchymal-to-epithelial transition (MET) at initiation stage of OSKM-induced reprogramming. Further analysis of gene expression and targets of Tfap2c during reprogramming by RNA-sequencing (RNA-seq) and ChIP-qPCR indicates that TFAP2C can promote epithelial gene expression by binding to their promoters directly. Finally, knockdown of E-cadherin (Cdh1), an important downstream target of TFAP2C and a critical regulator of MET antagonizes Tfap2c-mediated reprogramming. Taken together, we conclude that Tfap2c serves as a strong activator for somatic cell reprogramming through promoting the MET and inhibiting c-Myc-dependent apoptosis.

Hepatocellular carcinoma (HCC) is one of the most common and deadly malignant tumors in the world, especially in China. Follistatin‐like protein 5 (FSTL5) is a member of the FSTL family, which is involved in cell proliferation, migration, differentiation, and embryo development. We aimed to investigate the function and underlying mechanism of FSTL5 in HCC. FSTL5 expression was determined by immunohistochemistry staining in a liver cancer tissue microarray (TMA) and the correlation between FSTL5 and the prognosis of HCC patients was analysed. Further proliferation assay, colony formation assay, flow cytometry, and xenograft tumor model were performed to investigate the bioeffects of FSTL5 in HCC in vitro and in vivo. We found that FSTL5 expression was downregulated in HCC tissues and positively correlated with the prognosis of patients with HCC at tumor node metastasis stage I/II. Overexpression of FSTL5 efficiently impaired HCC growth both in vivo and in vitro with an exogenous manner. Mechanistic investigation demonstrated that FSTL5 promoted HCC cell apoptosis in a caspase‐dependent manner and regulated Bcl‐2 family proteins. These results indicate that FSTL5 may be a potential novel target for HCC treatment, and a biomarker for tumor prognosis.

Background Although the whole tumor cell vaccine can provide the best source of immunizing antigens, there is still a limitation that most tumors are not naturally immunogenic. Tumor cells genetically modified to secrete immune activating cytokines have been proved to be more immunogenic. IL-18 could augment proliferation of T cells and cytotoxicity of NK cells. GM-CSF could stimulate dendritic cells, macrophages and enhance presentation of tumor antigens. In our study, we used mouse GM-CSF combined with IL-18 to modify Lewis lung cancer LL/2, then investigated whether vaccination could suppress tumor growth and promote survival. Methods The Lewis lung cancer LL/2 was transfected with co-expressing mouse GM-CSF and IL-18 plasmid by cationic liposome, then irradiated with a sublethal dose X ray (100 Gy) to prepare vaccines. Mice were subcutaneously immunized with this inactivated vaccine and then inoculated with autologous LL/2 to estimate the antitumor efficacy. Results The studies reported here showed that LL/2 tumor cell vaccine modified by a co-expressing mouse GM-CSF and IL-18 plasmid could significantly inhibit tumor growth and increased survival of the mice bearing LL/2 tumor whether prophylactic or adoptive immunotherapy in vivo. A significant reduction of proliferation and increase of apoptosis were also observed in the tumor treated with vaccine of co-expressing GM-CSF and IL-18. The potent antitumor effect correlated with higher secretion levels of pro-inflammatory cytokines such as IL-18, GM-CSF, interferon-γ in serum, the proliferation of CD4+ IFN-γ+, CD8+ IFN-γ+ T lymphocytes in spleen and the infiltration of CD4+, CD8+ T in tumor. Furthermore, the mechanism of tumor-specific immune response was further proved by 51Cr cytotoxicity assay in vitro and depletion of CD4, CD8, NK immune cell subsets in vivo. The results suggested that the antitumor mechanism was mainly depended on CD4+, CD8+ T lymphocytes. Conclusions These results provide a new insight into therapeutic mechanisms of IL-18 plus GM-CSF modified tumor cell vaccine and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

SARI (suppressor of activator protein 1, regulated by IFN) functions as a tumour suppressor and is inactivated in various cancers. However, the mechanism underlying SARI inactivation in cancer remains elusive. In this study, we detected a high level of DNA methylation of the SARI promoter and an inverse correlation between SARI promoter methylation and expression in malignant tissues from patients with colon cancer. Furthermore, we found that the SARI promoter methylation status is a prognostic indicator for patients with colon cancer. A dCas9-multiGCN4/scFv-TET1CD-sgRNA-based SARI-targeted demethylation system (dCas9-multiGCN4/scFv-TET1CD-sgSARI) was constructed to precisely and specifically demethylate regions of SARI; this system resulted in the substantial activation of SARI expression. Further in vitro and in vivo data confirmed that dCas9-multiGCN4/scFv-TET1CD-sgSARI exerts anti-tumour effects by regulating tumour proliferation, apoptosis, and angiogenesis. Collectively, specific demethylation of the SARI promoter and restoration of endogenous SARI expression by dCas9-multiGCN4/scFv-TET1CD-SARI have therapeutic applications for colon cancer and perhaps for other cancers.

Class A scavenger receptor member 5 (SCARA5) is a new member of the Class A scavenger receptors that has been proposed recently as a novel candidate tumor suppressor gene in human hepatocellular carcinoma. In the present study, we found that SCARA5 expression was frequently downregulated in various cancer cell lines and tumor samples. In addition, upregulation of SCARA5 expression in human cancer cell line (U251) led to a significant decrease in cell proliferation, clone formation, migration, and invasion in vitro. Furthermore, systemic treatment of tumor‐bearing mice with SCARA5–cationic liposome complex not only reduced the growth of subcutaneous human glioma tumors, but also markedly suppressed the spontaneous formation of lung metastases. Similar results were obtained in another experiment using mice bearing experimental A549 lung metastases. Compared with the untreated control group, mice treated with SCARA5 exhibited reductions in both spontaneous U251 and experimental A549 lung metastases rates of 77.3% and 70.2%, respectively. Western blot analysis was used to explore the molecular mechanisms involved and revealed that SCARA5 physically associated with focal adhesion kinase. Interestingly, upregulation of SCARA5 inactivated signal transducer and activator of transcription 3, as well as downstream signaling including cyclinB1, cyclinD1, AKT, survivin, matrix metalloproteinase‐9 and vascular endothelial growth factor‐A. Overall, the findings of the present study provide the first evidence that SCARA5 might be a promising target for the development of new antimetastatic agents for the gene therapy of cancer.

It has been shown that Caspy2, a zebrafish active caspase, can efficiently suppress the growth of malignant tumor. The present study was designed to test whether combined gene therapy with IP-10, a potent antitumor chemokine, and Caspy2 would improve therapy efficacy. Recombinant plasmid expressing both Caspy2 and IP-10 genes was mixed with DOTAP-cholesterol nanoparticles. Immunocompetent mice bearing CT26 colon carcinoma, B16-F10 melanoma, and 4T1 breast carcinoma were treated with the complex. We found that the combined gene therapy more efficiently inhibited tumor growth, while efficiently prolonging the survival of tumor-bearing animals, compared with monotherapy. Moreover, a significant reduction in spontaneous lung metastasis could be observed in the 4T1 breast carcinoma model. Infiltration of CD8(+) T lymphocytes was also observed. In addition, apoptotic cells were widely detected by TUNEL assay and caspase-3 immunostaining in coadministered tumor tissues. The combination treatment also successfully inhibited angiogenesis and tumor cell proliferation as assessed by CD31 and Ki-67 immunostaining, respectively. Furthermore, depletion of CD8(+) T lymphocytes could significantly abrogate the antitumor activity, whereas the depletion of CD4(+) cells or natural killer cells showed partial abrogation. Rechallenged CT26 tumors were rejected in all of the surviving mice treated by combination therapy. Our results suggest that combined therapy with Caspy2 and IP-10 can significantly enhance antitumor activity by acting as an immune response initiator, apoptosis inducer, and angiogenesis inhibitor, which may be important for further applications in clinical cancer therapy.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is abundantly expressed in a variety of cancer cells, including lung cancer cells, resulting in low sensitivity of these cells to various apoptotic stimuli; Cisplatin (CDDP), a commonly used chemotherapeutic agent of several cancers, has a major limitation because of its toxicity at high concentration. In the present study, we constructed a plasmid encoding Survivin shRNA to knockdown survivin with low dose DDP both in vitro and in vivo. The specificity and potency of the shRNA were validated by western blot, flow cytometric and MTT in H292 lung cancers cells. In vivo, therapy experiments were conducted on nude mice bearing H292 xenograft tumors. The Survivin shRNA expression plasmid was administered systemically in combination with low-dose CDDP on a frequent basis. Assessments of angiogenesis, cell proliferation and apoptosis were performed by using immunohistochemistry against CD31, Ki67 and TUNEL assays, respectively. The results revealed that treatment with the Survivin shRNA plus low-dose CDDP reduced volume by approximately 83.13% compared with the blank control (P < 0.01), accompanied with angiogenesis inhibition (p < 0.01), tumor cell proliferation suppression (p < 0.05) and apoptosis induction (p < 0.01). Moreover, combination treatment also significantly reduced the mean tumor volume compared with other treatment alone (p < 0.05). Taken together, our study suggested that silencing of survivin sensitized H292 lung cancer cells to chemotherapy of CDDP, suggesting potential applications of the combined approach in the treatment of lung cancer.

Polo-like kinase 1 (Plk1) is a key cell cycle regulator that is frequently overexpressed in human hepatocellular carcinomas. Blockade of the Plk1 pathway has been reported to be capable of inducing anti-tumor effect. Here, plasmids containing U6 promoter-driven shRNAs against human Plk1 were constructed and transfected in human hepatocellular carcinoma cell line HepG2. ShRNA targeting Plk1 almost completely reduced Plk1 expression in HepG2 hepatocellular carcinoma cells, as confirmed by RT-PCR and Western blot. As a consequence, HepG2 cells exhibited reduced proliferation and enhanced apoptosis in vitro. Most importantly, Treatment with Plk shRNA-DOTAP:Chol complex significantly suppressed the growth of HepG2 xenografts, accompanied with phenotypic changes in tumor cells, including proliferation inhibition and apoptosis induction. Our study suggested that shRNA-mediated silencing of Plk1 might be a novel therapeutic approach against human hepatocellular carcinoma by inhibiting tumor cells proliferation and inducing apoptosis.

Recently, hPNAS-4 [human PNAS-4; also known as C1orf121 (Genbank accession number NP-057160)] was reported to be a novel pro-apoptotic protein in mammalian cells. However, at present there is little information about hPNAS-4 and its molecular mechanisms. In our present studies, the hPNAS-4 gene was first cloned into the pGEX-6p-1 vector with a GST (glutathione transferase) tag to express in soluble form in Escherichia coli induced with 0.5 mM IPTG (isopropyl beta-D-thiogalactoside) at 25 degrees C, and the recombinant hPNAS-4 was purified to near homogeneity with 96% purity by affinity chromatography and anion-exchange chromatography. The purified hPNAS-4 protein was further identified by liquid chromatography-ESI (electrospray ionization)-MS analysis and used to raise polyclonal antibodies that could specifically recognize the hPNAS-4 protein. In addition, hPNAS-4 is localized to the cytoplasm and the perinuclear compartment. Furthermore, the overexpression of hPNAS-4 in human A549 lung cancer cells resulted in decrease of cell viability via apoptosis. This study represented an important step to investigate the characterization for the new pro-apoptosis protein hPNAS-4, which should aid the discovery of new drug targets for the development of effective therapeutic approaches to cancer in the future.

PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. The objectives of this study were to determine whether PNAS-4 could enhance apoptosis induced by cisplatin besides its induction of apoptosis, and to evaluate the usefulness of combined treatment with mouse PNAS-4 (mPNAS-4) gene therapy and low-dose cisplatin chemotherapy in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models.In this study, the in vitro growth-inhibitory and pro-apoptotic effects of PNAS-4 and/or cisplatin on CT26, LL/2, and SKOV3 cancer cells were assessed by MTT assay, flow cytometric analysis, DNA fragmentation, and morphological analysis, respectively. The in vivo antitumor activity of combined treatment with mPNAS-4 gene therapy and low-dose cisplatin were evaluated in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models. Tumor volume and survival time were observed. Induction of apoptosis was also assessed in tumor tissues.In vitro, PNAS-4 inhibited proliferation of colon carcinoma (CT26), Lewis lung carcinoma (LL/2) and human ovarian cancer (SKOV3) cell lines via apoptosis, and significantly enhanced the apoptosis of CT26, LL/2, and SKOV3 cells induced by cisplatin. In vivo systemic administration of expression plasmid encoding mPNAS-4 (pcDNA3.1-mPS) and cisplatin, significantly decreased tumor growth through increased tumor cell apoptosis compared to treatment with mPNAS-4 or cisplatin alone.Our data suggests that the combined treatment with mPNAS-4 plus cisplatin may augment the induction of apoptosis in tumor cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis. The present study may provide a novel way to augment the antitumor efficacy of cytotoxic chemotherapy.

PNAS-4 has been demonstrated to induce apoptosis in U2OS cells. To evaluate its feasibility as a new strategy for cancer therapy, we analyzed its anti-tumor effect with or without gemcitabine in A549 lung cancer cells. MTT assay, Hoechst 33258 staining and flow cytometric analysis were used to determine the cytotoxicity of PNAS-4 alone or plus gemcitabine. The anti-tumor efficacy was further investigated in vivo with nude mice. PNAS-4 plasmid/liposome complexes were injected by tail vein every 4 days. Gemcitabine was given ip on a weekly schedule for 4 weeks. PNAS-4 alone and plus gemcitabine induced apoptosis in A549 cells in vitro. The xenograft lung cancer treated with PNAS-4 retarded growth compared with the empty vector. The combination of PNAS-4 with gemcitabine induced anti-tumor activity accompanied by an increase in apoptotic cells compared with PNAS-4 or gemcitabine alone. No other obvious toxicity was found. PNAS-4 therefore suppresses tumor growth in vivo and enhances sensitivity to gemcitabine. This suggests that the PNAS-4 gene could be a potential candidate for lung cancer therapy alone or in combination with gemcitabine.

Caspy2, a zebrafish protease, is an active caspase for inducing apoptosis in mammalian cells. To investigate whether caspy2‐mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid was mixed with cationic liposome and introduced into various tumour cell lines in vitro. Our data showed that caspy2 induced remarkable apoptosis of cancer cells in vitro. Treatment of mice‐bearing CT26 colon carcinoma or MethA fibrosarcoma with intratumoral injection of liposome‐caspy2 plasmid complex resulted in substantial killing of neoplastic cells and long‐term survivors. Apoptotic cells were widely distributed in caspy2‐treated tumour tissue. Infiltration of CD8(+) T lymphocyte was also observed apparently in the tumour tissue after the treatment with caspy2. Tumour‐specific major histocompatibility complex (MHC) class I‐dependent CD8(+) cytotoxic T lymphocyte activity was found by means of (51)Cr release assay. In MethA model, the mice acquired a long‐time protective immunity against the parental tumour cell re‐challenge. These results indicated that caspy2 can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumour effect. Furthermore, in vivo depletion of CD8(+) T lymphocytes could completely abrogate the antitumour immune activity, whereas the depletion of CD4(+) cells showed partial abrogation. In this study, we developed a novel anticancer strategy that combines both induction of apoptosis and immune response in mice‐bearing tumours with a single caspy2 gene. This approach may provide an important way for treatment of cancer.

Objective: The phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in ovarian cancer cell survival and proliferation. The aim of this study was to determine whether the suppression of the PI3K catalytic subunit p110αinhibits the growth of ovarian cancer cells in vitro and in vivo. Methods: The short hairpin RNA (shRNA) was used to knock down the expression of p110α in SKOV3 human ovarian cancer cells. The effects of shRNA on cell viability and apoptosis in vitro were assessed using the MTT assay and flow-cytometric analysis. Furthermore, the growth inhibition capacity of shRNA on ovarian carcinoma xenografts was tested in nude mice. Tumor cell proliferation, apoptosis and angiogenesis were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunohistochemistry, respectively. Results: Using sequence-specific shRNA, we have silenced the expression of p110α in SKOV3 cells. shRNA-mediated knockdown of p110α correlated in vitro with decreased cell viability and increased apoptosis. Furthermore, inhibition of p110α significantly delayed the growth of ovarian carcinoma xenografts, and ultimately resulted in decreased cell proliferation, induction of apoptosis as well as a reduction in microvessel density. Conclusions: Our findings suggest that shRNA-directed targeting of p110αraises the potential of its application in human ovarian cancer therapy.

Purpose: Tie-2 is an endothelium-specific receptor tyrosine kinase known to play a key role in tumor angiogenesis. The present study explores the feasibility of immunotherapy of tumors by using a protein vaccine based on chicken Tie-2 as a model antigen to break the immune tolerance against Tie-2 in a cross-reaction between the xenogeneic homologous and self-Tie-2.Experimental Design and Results: In this study, a chicken homologous Tie-2 protein vaccine (chTie-2) and a corresponding mouse Tie-2 vaccine as a control were prepared and the antitumor effect of these vaccines was tested in two tumor models (murine B16F10 melanoma and murine H22 hepatoma). Immunotherapy with chTie-2 was found effective in two tumor models. Autoantibodies against mouse Tie-2 were detected in sera of mice immunized with chTie-2 through Western blot analysis and ELISA assay. Anti-Tie-2 antibody-producing B cells were detectable by ELISPOT. Histologic examination revealed that autoantibodies were deposited on the endothelial cells of tumor tissues. Purified immunoglobulins from chTie-2-immunized mice could induce the apoptosis of human umbilical vein endothelial cells in vitro. Importantly, adoptive transfer of purified immunoglobulins led to antitumor effect in vivo; apparently, angiogenesis was significantly inhibited in these tumors. Furthermore, the antitumor activity and production of autoantibodies could be abrogated by depletion of CD4+ T lymphocytes.Conclusions: Our findings may provide a vaccine strategy for cancer therapy and show the potential utilization of interference with Tie-2 pathway.

Myeloid-derived suppressor cells (MDSCs) are known to play important roles in the development of immunosuppressive tumor microenvironment. A20 is a zinc-finger protein which could negatively regulate apoptosis in several cell types. However, the role of A20 in tumor microenvironment remains largely unknown. In this study, we found that A20 was over-expressed in MDSCs. The treatment of tumor-bearing mice with small interfering RNA targeting A20 (si-A20) inhibited the growth of tumors. The infiltration of MDSCs was dramatically reduced after si-A20 treatment, as compared to control groups, whereas the numbers of dendritic cells and macrophages were not affected. Also, injection of si-A20 improved T cell mediated tumor-specific immune response. Depletion of MDSCs with anti-Gr1 antibody showed similar antitumor effect and improved T cell response. TNF-α was highly expressed after si-A20 injection. Furthermore, si-A20 induced apoptosis of MDSCs in the presence of TNF-α both in vivo and in vitro. Cleaved Caspase-3 and Caspase-8 were elevated with the activation of JNK pathway after the induction of MDSC apoptosis by si-A20. Thus, our findings suggested that knockdown of A20 in tumor site inhibited tumor growth at least through inducing the apoptosis of MDSCs. A20 might be a potential target in anticancer therapy.

Most women with ovarian cancer are diagnosed at an advanced stage and there are few therapeutic options. Recently, monoclonal antibody therapies have had limited success, thus more effective antibodies are needed to improve long-term survival. In this report, we prepared polyclonal rabbit anti-ovarian cancer antibody (Poly Ab) by immunizing rabbits with the human ovarian cancer cell line SKOV3. The Poly Ab bound to SKOV3 and inhibited the cancer cells proliferation. Western blot analysis was conducted, which indicated that Poly Ab inhibited cancer cells through apoptosis involving the caspase signaling pathway including caspase-3 and caspase-9. Finally, compared with the control antibody, administration of Poly Ab reached 64% and 72% tumor inhibition in the subcutaneous and intraperitoneal xenograft mouse model, respectively. Our findings suggest that Poly Ab is an effective agent for apoptosis induction and may be useful as a safe anticancer agent for ovarian cancer therapy.

Aim Signal transducer and activator of transcription 3 (STAT3) plays an important role in the tumor formation, prognosis and chemoresistance of ovarian cancer. Our goal was to investigate the effect of silencing STAT3 on ovarian cancer cell apoptosis, proliferation, angiogenesis and expression of key targets in vitro and in vivo. Methods The ovarian cancer cell lines A2780CP and A2780s were used. STAT3 was knocked down by the plasmid-based short hairpin RNA (shRNA) expression system. In vitro, a colony formation assay and Hoechst staining were used to examine cell proliferation and apoptosis. The expression level of STAT3 and apoptosis-related proteins were determined by Western blot. The A2780CP intraperitoneal model was used to evaluate the effect of shSTAT3 on tumor growth in mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunostaining, respectively. Results Treatment with shSTAT3 resulted in apoptosis and inhibition of cell proliferation in vitro. Western blot analysis demonstrated that shSTAT3 induced the expression of cleaved caspase-3 and reduced the expression of survivin, Bcl-2 and vascular endothelial growth factor. In vivo, the tumor weight was reduced to 13.46% of 5% glucose by shSTAT3/lipoplexes (P

Currently, great interest is focused on the anti-neoplastic effects of CXC chemokine ligand 10 (IP-10/CXCL10). IP-10 has shown significant antitumor and anti-metastatic properties via immunological, antiangiogenic and anti-neoplastic mechanisms. However, very few studies on the antitumor activity of IP-10 in human ovarian cancer have been reported. The use of polymeric nanoparticles to deliver functional genes intraperitoneally holds much promise as an effective therapy for ovarian cancer. In our study, a recombinant plasmid expressing IP-10 (pVITRO-IP-10) was constructed, and biodegradable cationic heparin-polyethyleneimine (HPEI) nanogels were prepared to deliver pVITRO-IP-10 into SKOV3 human ovarian cancer cells. Transfection efficiency was detected by expression profiling of green fluorescent protein. The expression of IP-10 was determined using RT-PCR and western blot analysis. In vitro, cell proliferation was evaluated by MTT assay. Apoptosis was examined by Hoechst33258/PI staining and flow cytometry assays. The effect on the inhibition of angiogenesis was evaluated by tube formation assay using human umbilical vein endothelial cells (HUVECs). Moreover, a SKOV3 intraperitoneal ovarian carcinomatosis model was established to investigate the antitumor activity of HPEI+pVITRO-IP-10 complexes in nude mice. Tumor weights were evaluated during the treatment course. Cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay, and the antiangiogenic effect of pVITRO-IP-10 was assessed by CD31 immunochemical staining and alginate-encapsulated tumor cell assay. pVITRO-IP-10 was efficiently transfected into SKOV3 cells by HPEI nanogels. Intraperitoneal administration of HPEI+pVITRO-IP-10 complexes led to effective growth inhibition of ovarian cancer, in which tumor weight decreased by ~69.92% in the treatment group compared with that in the empty vector control group. Meanwhile, decreased cell proliferation, increased tumor cell apoptosis and reduction in angiogenesis were observed in the HPEI+pVITRO-IP-10 group compared with those in the control groups. These results indicated that HPEI nanogel delivery of pVITRO-IP-10 may be of value in the treatment against human ovarian cancer.

In the present study, we have used plasmid-based RNA interference (RNAi) strategy to downregulate the expression of epidermal growth factor receptor (EGFR) in EGFR wild-type (H292) and mutant (H1975) lung tumor models. The targeted knockdown of EGFR by small hairpin RNA not only inhibited growth of H292 xenograft but also inhibited H1975 lung cancer cell and xenograft, which bore L858R/T790M EGFR and was resistant to EGFR tyrosine kinase inhibitors. These data demonstrated that small hairpin RNA was an effective therapy against mutant EGFR-expressing cancer cells and thus considered to be a promising strategy in the treatment of lung cancers.

The HSulf-1 (heparan sulfate 6-O-endosulfatase 1) gene is an important element that modulates the sulfation status of heparan sulfate proteoglycans (HSPGs), leading to the interference of HSPG-related signal transduction pathways. HSulf-1 plays a key role in regulating cell proliferation, tumorigenesis and angiogenesis. Recently, some studies have reported that HSulf-1 is a down-regulated gene in the majority of examined tumor types. In our present study, a recombinant plasmid DNA carrying HSulf-1-cDNA (pHSulf-1) was constructed. The antitumor effect of pHSulf-1 delivered by heparin-polyethyleneimine (HPEI) nanogels on human ovarian cancer and the possible mechanisms of the antitumor efficacy in vivo were further investigated. Heparin-polyethyleneimine (HPEI) nanogels, as a new safe non-viral gene delivery carrier, were prepared to deliver the plasmid expressing HSulf-1 into HSulf-1-deficient SKOV3 human ovarian cancer cells in vitro and in vivo. pHSulf-1 could be efficiently transfected into SKOV3 ovarian cancer cells by HPEI nanogels in vitro and in vivo. Stable expression of HSulf-1 in vitro and in vivo was verified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Furthermore, a SKOV3 intraperitoneal ovarian carcinomatosis model was established to investigate the growth inhibition function of pHSulf-1 in nude mice. Tumor weight was measured. An anti-angiogenesis effect of pHSulf-1 in vivo was detected by CD31 immunostaining and alginate-encapsulate tumor cell assay. Assessment of apoptotic cells and proliferation index in tumor tissues were performed by TUNEL assay and Ki‑67 immunostaining. Intraperitoneal injection of pHSulf-1/HPEI complexes efficiently reduced tumor weight by approximately 87% compared with control groups (P

Polo-like kinase 1 (PLK1) showing a high expression in various kinds of tumors is considered a candidate target for cancer therapy. The aim of our study was to explore the effects of silencing PLK1 gene on human colorectal carcinoma cell line HCT-116 in vitro and in vivo. In vitro, the plasmids generating short hairpin RNA (shRNA)-targeting PLK1 were transfected into HCT-116 by using FugeneHD reagent, and the silencing potency was measured by RT-PCR, western blot, flow cytometry, and Caspase-Glo 3/7 assay, respectively. In vivo, the growth inhibition capacity of PLK1-shRNA on HCT-116 xenograft was measured in nude mice. Then, the silencing effect of PLK1 was analyzed by RT-PCR, western blot, and immunohistochemistry, respectively. Apoptosis, angiogenesis, and proliferation in tumor tissues were measured by TUNEL, CD31, and PCNA stain, respectively. The RNA interference targeting PLK1 significantly decreased the expression of PLK1 in vitro. More importantly, anti-PLK1 treatment in HCT-116 xenograft decreased tumor weight by 81.58% compared with the control group (p0.001), accompanied with decreased PLK1 mRNA and protein expression, increased cell apoptosis, and reduced angiogenesis and proliferation (p0.001). Our study showed that knockdown of PLK1 by shRNA might be the potential therapeutic approach against human colorectal carcinoma.

Purpose: VEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied in the cancer treatment. However, currently most of these anticancer drugs suffer some adverse effects. Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed. Experimental Design: In this investigation, we adopted a restricted de novo design method to design VEGFR2 inhibitors. We selected the most potent compound SKLB1002 and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro. Tumor xenografts in zebrafish and athymic mice were used to examine the in vivo activity of SKLB1002. Results: The use of the restricted de novo design method indeed led to a new potent VEGFR2 inhibitor, SKLB1002, which could significantly inhibit HUVEC proliferation, migration, invasion, and tube formation. Western blot analysis was conducted, which indicated that SKLB1002 inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal-regulated kinase, focal adhesion kinase, and Src. In vivo zebrafish model experiments showed that SKLB1002 remarkably blocked the formation of intersegmental vessels in zebrafish embryos. It was further found to inhibit a new microvasculature in zebrafish embryos induced by inoculated tumor cells. Finally, compared with the solvent control, administration of 100 mg/kg/d SKLB1002 reached more than 60% inhibition against human tumor xenografts in athymic mice. The antiangiogenic effect was indicated by CD31 immunohistochemical staining and alginate-encapsulated tumor cell assay. Conclusions: Our findings suggest that SKLB1002 inhibits angiogenesis and may be a potential drug candidate in anticancer therapy. Clin Cancer Res; 17(13); 4439–50. ©2011 AACR.

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P

Ovarian cancer is the number one cause of death from gynecologic malignancy. A defective p53 pathway is a hallmark of ovarian carcinoma. The p53 mutation correlates significantly with resistance to platinum-based chemotherapy, early relapse and shortened overall survival in ovarian cancer patients. PUMA (p53 upregulated modulator of apoptosis), a BH3-only Bcl-2 family protein, was recently identified as a transcriptional target of p53 and a potent apoptosis inducer in various cancer cells. In this study, we showed that the induction of PUMA by cisplatin was abolished in p53-deficient SKOV3 cells. Elevated expression of PUMA-induced apoptosis and sensitized A2780s and SKOV3 ovarian cancer cells to cisplatin, and the combination of PUMA and low-dose cisplatin, significantly suppressed xenograft tumor growth in vivo through enhanced induction of apoptosis compared with treatment with PUMA or cisplatin alone. The effects of PUMA were mediated by enhanced caspase activation and release of cytochrome c and Smac (second mitochondria-derived activator of caspase) into the cytosol. Furthermore, PUMA chemosensitized intrinsically resistant SKOV3 cells to cisplatin through downregulation of B-cell lymphoma-extra large (Bcl-x(L)) and myeloid cell leukemia sequence 1 (Mcl-1). PUMA-mediated Bcl-x(L) downregulation mainly happened at the transcription level, whereas PUMA-induced Mcl-1 down-regulation was associated with caspase-dependent cleavage and proteasome-mediated degradation. To our knowledge, these data suggest a new mechanism by which overexpression of PUMA enhances sensitivity of SKOV3 cells to cisplatin by lowering the threshold set simultaneously by Bcl-x(L) and Mcl-1. Taken together, our findings indicate that PUMA is an important modulator of therapeutic responses of ovarian cancer cells and is potentially useful as a chemosensitizer in ovarian cancer therapy.

Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we clonedXenopus laevisPNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in theXenopousembryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.

Purpose: Human neutrophil peptides (HNP1-3), small molecular antimicrobial peptides, are expressed within tumors and associated with tumor necrosis and inhibition of angiogenesis. Recent investigations have suggested that HNP1-3 are likely to be involved in the host immune responses to tumors.Experimental Design: We used recombinant pSec-HNP1, which expresses a secretable form of HNP1, to obtain expression of HNP1 in the tumor milieu in immunocompetent mice to explore the possible roles of HNP1 in tumor immunity. The antitumor effects were investigated in established CT26 colon cancer and 4T1 breast cancer models.Results: HNP1-mediated chemotactic and activating effects on immature dendritic cells were detected both in vitro and in vivo. Intratumoral expression of HNP1 resulted in not only significant tumor growth inhibition but also increased CTL infiltration within tumors. Adoptive transfer of splenocytes and a 51Cr release assay revealed specific cellular immune responses. Furthermore, increased antibodies were also found in sera from pSec-HNP1treated mice supporting specific humoral immune responses. Increased apoptosis and decreased angiogenesis were also shown in treated tumors.Conclusions: These findings indicate that HNP1 can exert multiple antitumor effects through different mechanisms; more importantly, HNP1 mediates host immune responses to tumors in situ through the recruitment and subsequent activation of immature dendritic cells and thus shows promising potential in cancer therapy. (Clin Cancer Res 2009;15(22):690111)

PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. Recent studies have indicated that honokiol can induce apoptosis, inhibit angiogenesis, and suppress tumor growth. In the present study, we investigated whether mouse PNAS-4 (mPNAS-4) could augment the apoptosis of tumor cells induced by honokiol in vitro, and whether the antiangiogenic activity of honokiol and induction of apoptosis by mPNAS-4 could work cooperatively to improve the antitumor efficacy in vivo. In vitro, mPNAS-4 inhibited proliferation of murine colorectal carcinoma CT26 and Lewis lung carcinoma LL2 cells through induction of apoptosis, and significantly augmented the apoptosis of CT26 and LL2 cells induced by honokiol. Compared with treatment with mPNAS-4 or honokiol alone, in vivo systemic administration of an expression plasmid encoding mPNAS-4 and low-dose honokiol significantly suppressed tumor growth through the enhanced induction of apoptosis and the augmented inhibition of angiogenesis. Our data suggest that the combined treatment with mPNAS-4 plus honokiol augments antitumor effects in vitro and in vivo, and that the improved antitumor activity in vivo may be associated with enhanced induction of apoptosis and augmented inhibition of angiogenesis. The present study may provide a novel and effective method for the treatment of cancer.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase implicated in cancer cell survival, proliferation, and in various steps in the metastatic cascade. In the present study, we took advantage of a cationic liposome as gene carrier and targeted FAK function through both in vitro and in vivo approaches. We utilized a plasmid-encoded hairpin RNA targeting the human FAK mRNA (pGensil2-shRNA/FAK), as a means to inhibit FAK expression for evaluating its anti-tumor effect in vitro and in vivo. Human MDA-MB-435S breast cancer cells were transfected with pGensil2-shRNA/FAK and examined for apoptosis by propidium iodide staining, DNA ladder, and flow cytometric analysis. For in vivo study, subcutaneous breast carcinomatosis models in nude mice were established to evaluate the therapeutic potential of pGensil2-shRNA/FAK. Assessments of proliferation (Ki-67), apoptosis (TUNEL) and angiogenesis (CD31) were done using immunohistochemical analysis. Transcripts expressed from plasmid both in vitro and in vivo were identified by northern blot analysis. pGensil2-shRNA/FAK effectively down-regulated the expression of FAK as demonstrated in vitro by real time RT-PCR and western blot analysis, whereas by real time RT-PCR and IHC staining of MDA-MB-435S tumors growing subcutaneously. Breast cancer cells lacking FAK expression undergo apoptosis in vitro. Systemic delivery of cationic liposome-complexed plasmids targeting FAK, resulted in the diminishment of subcutaneous tumor growth beyond the effects observed with liposomes carrying a non-specific shRNA. This diminishment in growth was associated with elevated levels of apoptosis (TUNEL staining), decreased cell proliferation (Ki-67 staining) and diminished endothelial cell density (CD31 staining). These results indicate that the systemic delivery of plasmid DNA targeting FAK function using cationic liposome as a gene carrier, represents a promising avenue for breast cancer therapy.

We transfected ovarian cancer cells and administered recombinant plasmid encoding hPNAS-4 to nude mice bearing ovarian cancer, aiming to evaluate the effect of hPNAS-4 against ovarian cancer in vitro and in vivo.Ovarian cancer SKOV3 cells were transfected with hPNAS-4-plasmid, and cell proliferation was evaluated by MTT assay; apoptosis was examined by DNA ladder, Hoechst33258 staining and flow-cytometric assays. Nude mice bearing ovarian cancers were treated with hPNAS-4-p/liposome. Tumor growth was determined and survival was recorded. TUNEL assay and microvessel density was assessed to evaluate apoptosis and angiogenesis.Both inhibition of proliferation (p0.05) and induction of apoptosis (p0.05) were observed in SKOV3 cells transfected with hPNAS-4-p in vitro. In hPNAS-4-p-treated tumor cells in vivo, tumor growth significantly decreased, while the survival time of tumor-bearing mice was prolonged compared with control groups (p0.05). Increased apoptosis of tumor cells and decreased angiogenesis in tumor tissue were also observed.Our promising results on the potential antitumor effects of hPNAS-4 on ovarian cancer in vitro and in vivo may be explained, in part, by the induction of apoptosis and inhibition of angiogenesis. Consequently, hPNAS-4 has potential as a new gene therapy for human ovarian cancer.

Angiogenesis is critical to the growth and metastasis of solid tumors, and acquired drug resistance is one of the major hindrances to chemotherapy. Thus, we sought a rational strategy using the combination of antiangiogenic biotherapy and chemotherapy for cancer therapy. We explored the efficacy of a strategy combining low-dose cisplatin and a recombinant xenogeneic endoglin as a protein vaccine, which we previously demonstrated to have effective antiangiogenic effects in several mouse models. We found that both low-dosage cisplatin and xenogeneic endoglin vaccine individually resulted in effective suppression of tumor growth in 2 tumor models via inhibition of tumor angiogenesis. Remarkably, the combination therapy resulted in not only significant antiangiogenic effects but also additional promotion of tumor cell apoptosis and inhibition of tumor cell proliferation, without any ensuing increase in host toxicity during the course of treatment, which lasted for 6 months. In addition, the combination demonstrated a synergistic relationship, which was shown in all of the synergistic indexes, i.e., tumor volume, angiogenesis, apoptosis and proliferation. Both antibodies and antibody-producing B cells against mouse self-endoglin were observed in all mice immunized by the xenogeneic endoglin vaccine (alone and combination), which suggested that low-dose cisplatin did not suppress the host immune response but potentiated the antitumor activity of the xenogeneic endoglin vaccine. These observations may provide the basis for an effective alternative strategy for cancer therapy in the near future.

Vesicular stomatitis virus (VSV) has been shown to replicate rapidly in vitro and kill selectively a variety of tumor cell lines. The present study was designed to determine whether gemcitabine potentiates the antitumor activity of VSV in vitro and in vivo. A549 human lung adenocarcinoma cells and LLC Lewis lung carcinoma cells were treated with VSV (0.1-10 plaque-forming units per cell) plus gemcitabine (20 nM to 20 microM). Mice bearing A549 or LLC were treated with VSV (5 x 10(4) to 1 x 10(8) plaque-forming units) daily for 5 days plus gemcitabine (5-125 mg/kg/day) once every 3 days for 4 times. Induction of apoptosis and effects on growth inhibition were assessed. The lung cancer cells treated with VSV plus gemcitabine displayed the apparently increased apoptotic cells compared with treatment with VSV or gemcitabine alone. The combined treatment with VSV plus gemcitabine induced the apparent antitumor activity with complete regression of the established lung cancer in both A549 and LLC lung cancer models and augmented the induction of apoptosis in lung cancer cells in vivo as well. This study suggests that the combined treatment with VSV plus gemcitabine may augment the induction of apoptosis in lung cancer cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis in lung cancer cells. The present findings may be of importance to the further exploration of the potential application of this combined approach in the treatment of lung cancer.

Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.