Broad-spectrum anti-tumor and anti-metastatic DNA vaccine based on p62-encoding vector

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors – B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.     

Plasmid DNA-coding p62 as a bone effective anti-inflammatory/anabolic agent

We recently reported that a DNA plasmid coding p62-SQSTM1 acts as an effective anti tumor vaccine against both transplantable mouse tumors and canine spontaneous mammary neoplasms. Here we report the unexpected finding that intramuscular delivery of p62 DNA exerts a powerful anti-osteoporotic activity in a mouse model of inflammatory bone loss (i.e, ovariectomy) by combining bone-sparing and osteo-synthetic effects. Notably, the suppression of osteoporosis by p62DNA was associated with a sharp down-regulation of master inflammatory cytokines, and up-regulation of endogenous p62 protein by bone-marrow stromal cells. The present data provide a solid rational to apply p62 DNA vaccine as a safe, new therapeutic for treatment of inflammatory related bone loss diseases.     

The antimicrobial peptide pardaxin exerts potent anti-tumor activity against canine perianal gland adenoma

Pardaxin is an antimicrobial peptide of 33 amino acids, originally isolated from marine fish. We previously demonstrated that pardaxin has anti-tumor activity against murine fibrosarcoma, both in vitro and in vivo. In this study, we examined the anti-tumor activity, toxicity profile, and maximally-tolerated dose of pardaxin treatment in dogs with different types of refractory tumor. Local injection of pardaxin resulted in a significant reduction of perianal gland adenoma growth between 28 and 38 days post-treatment. Surgical resection of canine histiocytomas revealed large areas of ulceration, suggesting that pardaxin acts like a lytic peptide. Pardaxin treatment was not associated with significant variations in blood biochemical parameters or secretion of immune-related proteins. Our findings indicate that pardaxin has strong therapeutic potential for treating perianal gland adenomas in dogs. These data justify the veterinary application of pardaxin, and also provide invaluable information for veterinary medicine and future human clinical trials.     

Combinatory anti-tumor effects of electroporation-mediated chemotherapy and wild-type p53 gene transfer to human esophageal cancer cells

Delivery of electric pulses to an established solid tumor augments the permeability of cell membrane and increases the susceptibility of tumors to an anti-cancer agent that is administered in the vicinity of tumors. Forced expression of the wild-type p53 gene in tumor cells that have non-functional p53 gene(s) can also enhance their sensitivity to a DNA-damaging agent. To investigate the feasibility of electroporation-mediated therapy for cancer, electric pulses were delivered to human esophageal tumors developed in nude mice after they received an anti-cancer agent and/or plasmid DNA containing the wild-type p53 gene. The growth of esophageal tumors was suppressed with electroporation-mediated chemotherapy compared with the treatment with an anti-cancer agent or electroporation alone. Intratumoral injection of the wild-type p53 gene into p53-mutated esophageal tumors followed by electroporation also inhibited tumor growth. When mice were administered with the wild-type p53 gene and an anti-cancer agent, subsequent electroporation produced a synergistic therapeutic effect. Combinatory transfer of plasmid DNA and a pharmacological agent by electroporation is thereby a possible therapeutic strategy for the treatment of solid tumors.     

The role of the p53 molecule in cancer therapies with radiation and/or hyperthermia

In recent years, cancer-related genes have been analyzed as predictive indicators for cancer therapies. Among those genes, the gene product of a tumor suppressor gene p53 plays an important role in cancer therapy, because the p53 molecule induces cell-cycle arrest, apoptosis and depression of DNA repair after cancer therapies such as radiation, hyperthermia and anti-cancer agents. An abnormality of the p53 gene might introduce low efficiency in their cancer therapies. Mutations of p53 are observed at a high frequency in human tumors, and are recognized in about half of all malignant tumors in human. In the both systems of a human cell culture and their transplanted tumor, the sensitivities to radiation, heat and anti-cancer agents were observed in wild-type p53 cells, but not in mutated or deleted p53 cells. In this review, we discuss the p53 activation signaling pathways through the modification of p53 molecules such as phosphorylation after radiation and/or hyperthermia treatments.     

Proton pump inhibitors for the treatment of cancer in companion animals

The treatment of cancer presents a clinical challenge both in human and veterinary medicine. Chemotherapy protocols require the use of toxic drugs that are not always specific, do not selectively target cancerous cells thus resulting in many side effects. A recent therapeutic approach takes advantage of the altered acidity of the tumour microenvironment by using proton pump inhibitors (PPIs) to block the hydrogen transport out of the cell. The alteration of the extracellular pH kills tumour cells, reverses drug resistance, and reduces cancer metastasis. Human clinical trials have prompted to consider this as a viable and safe option for the treatment of cancer in companion animals. Preliminary animal studies suggest that the same positive outcome could be achievable. The purpose of this review is to support investigations into the use of PPIs for cancer treatment cancer in companion animals by considering the evidence available in both human and veterinary medicine.     

Efficient electrotransfection into canine muscle

Two different types of electroporation protocols have been developed for efficient electrotransfer of plasmid DNA into skeletal muscle of experimental animals. At first, only low voltage electric pulses have been used, but lately, a combination of high and low voltage pulses has been suggested as more efficient. Up to date, in dogs, this type of electroporation protocol has never been used for muscle targeted plasmid DNA electrotransfection. In this study, we used two different DNA plasmids, one encoding green fluorescent protein and one encoding human interleukin-12. Five different electroporation protocols were evaluated. Three of them featured different combinations of high and low voltage pulses, and two were performed with delivery of low voltage pulses only. Our study shows that combination of 1 high voltage pulse (600 V/cm, 100 mus), followed by 4 low voltage pulses (80 V/cm, 100 ms, 1 Hz) yielded in the same transfection efficiency as the standard trains of low voltage pulses. However, this protocol is performed quicker and, thus, more suitable for potential use in clinical practice. In addition, it yielded in detectable systemic expression of human interleukin-12. Electrotransfer of either of the plasmids was associated with only mild and transitory local side effects, without clinically detectable systemic side effects. The results indicate that electrotransfection is a feasible, effective, and safe method for muscle targeted gene therapy in dogs, which could have potential for clinical applications in veterinary medicine of small animals.     

CpG寡聚脱氧核苷酸在肿瘤免疫治疗中的应用进展

CpG寡聚脱氧核苷酸(oligodeoxynucleotide,ODN)具有激活机体免疫系统的作用.CpG ODN能够直接诱导浆细胞样树突状细胞(plasmacytoid dendritic cell,pDC)的活化和成熟,促进Th1型细胞因子的分泌和B细胞分化成浆细胞.CpG ODN作为肿瘤疫苗的佐剂已应用于多个临床试验中.单独应用CpG ODN具有抗肿瘤作用,联合其他抗肿瘤治疗(如单克隆抗体、化学治疗、放射治疗和细胞因子等)具有协同抗肿瘤作用.虽然在应用CpG ODN治疗ⅢB~Ⅳ期非小细胞肺癌(non-small cell lung cancer,NSCLC)的Ⅲ期临床试验中,与标准化疗方案相比没有延长患者的中位生存期并发生较严重的不良反应,但是在其他临床试验中CpG ODN具有明确的抗肿瘤作用.CpG ODN在抗肿瘤治疗中的有效性和安全性方面还需要进一步的临床试验证实.     

RAPD differentiation of Malassezia spp. from cattle, dogs and humans

The molecular characterization of Malassezia spp. isolates from animals and humans has not been thoroughly studied. We have analysed the DNA profile by random amplified polymorphic DNA (RAPD)–PCR to compare the genetic diversity between isolates from the external ears of cattle, dogs and humans. The analysis of electrophoretic profiles on 8% polyacrylamide gel and their phenograms showed genetic heterogeneity between RAPD profiles of Malassezia furfur and Malassezia slooffiae isolates from humans and cattle and between Malassezia pachydermatis isolates from dogs and cattle. Intra-species variations in DNA pattern of Malassezia isolates and the presence of specific genetic types in cattle, dogs or humans were observed. A review of genetic heterogeneity of these yeast in veterinary and human medicine studies is given considering a possible transmission animal to human or human to animal. Additional studies must clarify the differences between the RAPD band patterns observed in this and other studies, which would facilitate monitoring of Malassezia spp. carriage in domestic animals and in humans.     

Pulling the plug on a cancer cell by eliminating XIAP with AEG35156

XIAP, the X-linked inhibitor of apoptosis, is the best example of an endogenous cellular suppressor of apoptosis. XIAP is effective because it directly limits the activity of several critical death-inducing caspases, notably caspase-3, -7 and -9, either by direct enzyme inhibition or through ubiquitin-mediated proteasomal degradation. Furthermore, XIAP acts simultaneously at several nodes in the apoptotic cascade, blocking both the intrinsic and extrinsic death pathways, and thereby preventing feed-forward amplification loops that would otherwise lead to cell death. XIAP over-expression, or increased activity, is associated with cancer progression, resistance to therapy and poor prognosis. Targeting XIAP gene expression by antisense oligonucleotides, or other approaches, demonstrates anti-cancer effects with XIAP down-regulation. These early preclinical studies led to the development of a clinical candidate mixed-backbone antisense oligonucleotide, AEG35156, against XIAP for the treatment of cancer. Published clinical results for the first-in-class and first-in-human trials of AEG35156 are summarized herein, including single agent and combination chemotherapy phase-I or -II trials for solid tumors, lymphoma, and acute myeloid leukemia. These trials demonstrate the safety of AEG35156, as well as some initial promising signs of anti-cancer activity.     

Hydroxycamptothecin-loaded Fe3O4 nanoparticles induce human lung cancer cell apoptosis through caspase-8 pathway activation and disrupt tight junctions

10-Hydroxycamptothecin (HCPT) elicits strong anti-cancer effects and is less toxic than camptothecin (CPT), making it widely used in recent clinical trials. However, its low solubility limits its application as an effective anti-cancer therapy. In the present study we investigate the hypothesis that the unique water dispersible oleic acid-Triton X-100-coated Fe3O4 nanoparticles loaded with HCPT disrupt epithelial cell-cell junctions and induce human lung cancer cell apoptosis through the caspase-8 pathway. We characterized the HCPT-loaded nanoparticles and determined their effects on lung cancer cell viability and apoptosis by using immunofluorescence light microscopy and SDS-PAGE/immunoblots. We found that HCPT-loaded nanoparticles elicited an anti-proliferative effect in a dose-dependent manner. HCPT-loaded nanoparticles reduced the expression of cell-cell junction protein claudins, E-cadherin and ZO-1, and transmission electron microcopy demonstrated a disrupted tight junction ultrastructure. Transepithelial electric resistance was also reduced, indicating the reduction of tight junction functions. The HCPT-loaded nanoparticles increased phosphorylation of p38 and SAPK/JNK while it showed no effects on p42/44 MAP kinase. Compared with void Fe3O4 nanoparticles or HCPT drug alone, HCPT drug-loaded nanoparticles evoked synergistic effects by increasing cell apoptosis with enhanced activation of the caspase-8 pathway. Therefore, our current study highlights the potential of HCPT drug-loaded nanoparticles as a chemotherapeutic agent for increasing anti-cancer drug efficacy.     

Autologous versus allogeneic cell-based vaccines?

Devitalized tumor cells either autologous or allogeneic have been used as anti-cancer vaccines with the purpose of facilitating the induction of an immune response able to destroy growing tumor cells since the identification of tumor antigens was deemed not to be necessary, particularly in the autologous system. Such vaccines were tested first in animal models and then in the clinics as unmodified tumor cells or after insertion of genes coding for factors known to increase the immune response against tumors. These vaccines were usually given by subcutaneous injections along with different immunological adjuvants. Such immunization approaches were found to be effective in mice when carried out in a tumor preventive setting but significantly less in the therapeutic context, that is, in the presence of an established tumor. By analyzing several clinical trials of vaccination using either autologous or allogeneic unmodified and gene-modified tumor cells published in the last 10 to 15 years, we conclude for a lack of sufficient evidence for efficacy of this strategy in inducing both a strong immune response and a therapeutic response. A potential variant of this strategy is the direct intratumoral injection of immunostimulatory genes delivered by vectors in vivo. But even this approach failed to provide a statistically significant clinical benefit for the cancer patients.We also point out the inherent drawbacks of the tumor cell-based vaccine strategy that include (a) a limited frequency by which human tumor lines can be obtained from clinical samples, (b) the low number of available cells for vaccination, (c) the release of immune-suppressive factors by tumor cells, and (d) the cost and time necessary for standardization and collecting/expanding a number of cells according to the approved regulatory requirements. Thus, taking into consideration the new developments in cancer vaccines, we believe that tumor cell-based vaccines should be dismissed as anti-cancer vaccines unless a clear benefit could be demonstrated by the few ongoing trials of combination with new immunomodulating reagents (eg, anti-CTLA4, PD-1, chemotherapy).     

Microbial-based therapy of cancer: a new twist to age old practice

The use of bacteria in the regression of tumors has long been known. Various approaches for using bacteria in cancer therapy include the use of bacteria as sensitizing agents for chemotherapy, as delivery agents for cancer drugs and as agents for gene therapy. The tumor regression stimulated by infecting microorganisms has been attributed to activation of the immune system of the host. However, recent studies indicate that when tumor-harboring mice with defective immune systems are infected with certain microorganisms, the regression of the tumor is still observed, suggesting that there are other host factors contributing to the microbial associated regression of tumors. Since the use of live or attenuated bacteria for tumor regression has associated toxic effects, studies are in progress to identify a pure microbial metabolite or any component of the microbial cell that might have anti-cancer activity. It has now been demonstrated that a redox protein from Pseudomonas aeruginosa, a cupredoxin, can enter into human cancer cells and trigger the apoptotic cell death. In vivo, this cupredoxin can lead to the regression of tumor growth in immunodeficient mice harboring xenografted melanomas and breast cancer tumors without inducing significant toxic effects, suggesting that it has potential anti-cancer activity. This bacterial protein interacts with p53 and modulates mammalian cellular activity. Hence, it could potentially be used as an anti-cancer agent for solid tumors and has translational value in tumor-targeted or in combinational-biochemotherapy strategies for cancer treatments. Here, we focus on diverse approaches to cancer biotherapy, including bacteriolytic and bacterially-derived anti-cancer agents with an emphasis on their mechanism of action and therapeutic potential.     

The emerging role of DNA repair proteins as predictive, prognostic and therapeutic targets in cancer

Advanced cancer is the second leading cause of death in the western world. Chemotherapy and radiation are the two main treatment modalities currently available to improve patient outcomes, but treatment related toxicity and the emergence of resistance limit their effectiveness. Hence there is an urgent need to develop novel treatment strategies. Rapid advances in cancer biology have identified key pathways involved in the repair of DNA damage induced by chemotherapeutic agents and irradiation. Efficient DNA repair in the cancer cell is an important mechanism for therapeutic resistance. Up to 130 genes have been identified that are associated with human DNA repair. Several of these proteins are emerging as important predictive and prognostic factors in solid tumours. Inhibition of DNA repair has the potential to enhance the efficacy of currently available DNA damaging agents. In recent years, several promising drug targets have been identified and novel drugs synthesised that target specific DNA repair proteins. These agents have shown impressive anti-cancer effects in preclinical studies in combination with chemotherapy or irradiation. Their role in human cancer is now being investigated in early phase clinical trials in combination with chemotherapy. MGMT inhibitors, PARP inhibitors and methoxyamine are currently in early stages of clinical development. Innovative clinical trial designs are essential to evaluate the potential of DNA repair inhibitor in cancer therapy.     

Pretreatment with cisplatin enhances E7-specific CD8+ T-Cell-mediated antitumor immunity induced by DNA vaccination.

PURPOSE: Because the combination of multiple modalities for cancer treatment is more likely to generate more potent therapeutic effects for the control of cancer, we have explored the combination of chemotherapy using cisplatin, which is routinely used in chemotherapy for advanced cervical cancer, with immunotherapy using DNA vaccines encoding calreticulin (CRT) linked to human papillomavirus type 16 E7 antigen (CRT/E7) in a preclinical model. EXPERIMENTAL DESIGN: We characterized the combination of cisplatin with CRT/E7 DNA vaccine using different regimen for its potential ability to generate E7-specific CD8+ T-cell immune responses as well as antitumor effects against E7-expressing tumors. RESULTS: Our results indicate that treatment of tumor-bearing mice with chemoimmunotherapy combining cisplatin followed by CRT/E7 DNA generated the highest E7-specific CD8+ T-cell immune response and produced the greatest antitumor effects and long-term survival as well as significant levels of E7-specific tumor-infiltrating lymphocytes compared with all the other treatment regimens. Furthermore, we found that treatment with cisplatin leads to the cell-mediated lysis of E7-expressing tumor cells in vitro and increased number of E7-specific CD8+ T-cell precursors in tumor-bearing mice. In addition, we observed that E7-specific CD8+ T cells migrate to and proliferate in the location of TC-1 tumors in mice treated with cisplatin. CONCLUSIONS: Thus, our data suggest that chemoimmunotherapy using cisplatin followed by CRT/E7 DNA vaccine is an effective treatment against E7-expressing tumors and may potentially be translated into the clinical arena.     

Irradiation-hyperthermia in canine hemangiopericytomas: large-animal model for therapeutic response

Results of irradiation-hyperthermia treatment in 11 dogs with naturally occurring hemangiopericytoma were reported. Similarities of canine and human hemangiopericytomas were described. Orthovoltage X-irradiation followed by microwave-induced hyperthermia resulted in a 91% objective response rate. A statistical procedure was given to evaluate quantitatively the clinical behavior of locally invasive, nonmetastatic tumors in dogs that were undergoing therapy for control of local disease. The procedure used a small sample size and demonstrated distribution of the data on a scaled response as well as transformation of the data through classical parametric and nonparametric statistical methods. These statistical methods set confidence limits on the population mean and placed tolerance limits on a population percentage. Application of the statistical methods to human and animal clinical trials was apparent.     

重组人血管内皮抑制素与放射治疗敏感性的相关性研究进展

随着抗血管生成治疗恶性肿瘤理论的提出,重组人血管内皮抑素（恩度）作为一种新型的抗肿瘤药物,与传统的放疗联合具有协同作用,在基础及临床研究中均证实了恩度的放疗增敏作用。可能机制包括改善肿瘤内乏氧情况,作用于血管内皮细胞,调整细胞周期和调节促血管生成因子水平等。将恩度与放疗联合有望成为一种新的治疗策略应用于临床,提高恶性肿瘤的治疗效果。     

Activation of p53/p21Waf1/Cip1 pathway by 5-aza-2'-deoxycytidine inhibits cell proliferation, induces pro-apoptotic genes and mitogen-activated protein kinases in human prostate cancer cells

The tumor suppressor gene p53 plays an essential role in cell proliferation and apoptosis. Due to its relevance to cancer therapy, most studies have focused on the cellular consequences of p53 activation in relation to cytotoxic drugs. 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anti-cancer drug for the treatment of leukemia and solid tumors. However, the mechanism by which 5-aza-CdR exerts its anti-neoplastic activity remains unclear. Here, we address the role of p53 in regulating cellular responses to 5-aza-CdR treatment in human prostate cancer cells. We found that 5-aza-CdR induces p53 and p21Waf1/Cip1 expression associated with inhibition of cell proliferation in LNCaP cells (p53 wild-type), but not in DU145 cells (p53 mutant). By using pifithrin-alpha, a chemical inhibitor of p53, we confirmed that the increase in p21Waf1/Cip1 expression and inhibition of cell proliferation in LNCaP cells by 5-aza-CdR is p53-dependent. Also, the activation of p53 and p21Waf1/Cip1 pathway by 5-aza-CdR modified multiple gene expressions including apoptotic target genes and MAP kinases in LNCaP cells. 5-aza-CdR-induced apoptosis in LNCaP cells is assessed by DNA fragmentation analysis. Furthermore, knockdown of p53 by pU6-p53 siRNA vector suggests the involvement of MAP kinases in the process of 5-aza-CdR-mediated activation of p53 pathway to inhibit cell proliferation and induce apoptosis. Finally, the comet or SCGE assay and methylation-sensitive restriction analysis demonstrated that 5-aza-CdR induced p53 and p21Waf1/Cip1 expression as a consequence of DNA damage and independent of DNA demethylation. Our findings suggest that 5-aza-CdR induces anti-neoplastic activity primarily through the activation of p53 pathway in response to DNA damage and subsequently leads to inhibition of cell proliferation as well as induction of apoptosis. Therefore, our data indicate that p53 status in tumor cells may be critical for the clinical efficacy and toxicity of 5-aza-CdR.