Immunogenic Cell Death Enhances Immunotherapy of Diffuse Intrinsic Pontine Glioma: From Preclinical to Clinical Studies.

Diffuse intrinsic pontine glioma (DIPG) is the most lethal tumor involving the pediatric central nervous system. The median survival of children that are diagnosed with DIPG is only 9 to 11 months. More than 200 clinical trials have failed to increase the survival outcomes using conventional cytotoxic or myeloablative chemotherapy. Immunotherapy presents exciting therapeutic opportunities against DIPG that is characterized by unique and heterogeneous features. However, the non-inflammatory DIPG microenvironment greatly limits the role of immunotherapy in DIPG. Encouragingly, the induction of immunogenic cell death, accompanied by the release of damage-associated molecular patterns (DAMPs) shows satisfactory efficacy of immune stimulation and antitumor strategies. This review dwells on the dilemma and advances in immunotherapy for DIPG, and the potential efficacy of immunogenic cell death (ICD) in the immunotherapy of DIPG.

Endoscopic retrograde appendicitis therapy versus laparoscopic appendectomy versus open appendectomy for acute appendicitis: a pilot study.

BACKGROUND: An increasing number of studies have shown the merits of endoscopic retrograde appendicitis therapy (ERAT) in diagnosing and treating acute uncomplicated appendicitis. However, no related prospective controlled studies have been reported yet. Our aim is to assess the feasibility and safety of ERAT in the treatment of acute uncomplicated appendicitis. METHODS: In this open-label, randomized trial, participants were randomly allocated to the ERAT group, laparoscopic appendectomy (LA) group and open appendectomy (OA) group. The primary outcome was the clinical success rate of the treatment. Intention-to-treat analysis was used in the study. RESULTS: The study comprised of 99 patients, with 33 participants in each group. The clinical success rate was 87.88% (29/33), 96.97% (32/33) and 100% (33/33) in the ERAT, LA and OA group, respectively. In the ERAT group, 4 patients failed ERAT due to difficult cannulation. In LA group, 1 patient failed because of abdominal adhesion. There were no significant differences among the three treatment groups regarding the clinical success rate (P = 0.123). The median duration of follow-up was 22 months. There were no significant differences (P = 0.693) among the three groups in terms of adverse events and the final crossover rate of ERAT to surgery was 21.21% (7/33). CONCLUSION: ERAT can serve as an alternative and efficient method to treat acute uncomplicated appendicitis. Trial registration The study is registered with the WHO Primary Registry-Chinese Clinical Trial Registry (ChiCTR1900025812).

DCA increases the antitumor effects of capecitabine in a mouse B16 melanoma allograft and a human non-small cell lung cancer A549 xenograft.

PURPOSE: Capecitabine is one of the few chemotherapy drugs with high oral availability. Recently, sodium dichloroacetate (DCA) has shown great potential as an anticancer agent. In the present study, we assessed the anticancer effect of DCA in combination with capecitabine for cancers that modestly expressed TP. METHODS: A mouse B16 melanoma allograft and a human non-small cell lung cancer A549 xenograft were used to assess the effect of DCA and capecitabine combined treatment. Histology and immunohistochemistry were used to detect the apoptosis and proliferation of cancer cells. Real-time PCR and Western blot were carried out to detect the expression of TP and caspases, respectively. RESULTS: For the first time, we report that DCA increased the antitumor effects of capecitabine in a mouse B16 allograft and a human A549 xenograft by promoting apoptosis of tumor cells. DCA has little effect on the expression of TP. CONCLUSIONS: Our finding suggests that DCA in combination with capecitabine might be potential as a new therapeutic regimen against some cancers.

Ferritin light chain and squamous cell carcinoma antigen 1 are coreceptors for cellular attachment and entry of hepatitis B virus.

Overexpression of squamous cell carcinoma antigen 1 (SCCA1) in hepatitis G2 (HepG2) and Chinese hamster ovary cells can increase hepatitis B virus (HBV) binding capacity by interacting with the preS1 domain of the HBV surface antigen. However, the magnitude of increase in binding capacity was higher by several orders in the former, indicating the existence of additional factor(s) produced by HepG2 cells, which facilitates HBV attachment. Ferritin light chain (FTL) was identified as the sole high hit candidate by screening human liver cDNA library using a bacterial two-hybrid system with either preS or SCCA1 as the bait. Subsequent in vitro protein-protein interaction assays confirmed the binding activity of FTL to both preS and SCCA1, as well as the formation of triple complex preS-FTL-SCCA1, and narrowed down the binding sites on FTL. In vitro overexpression of FTL could further enhance HBV attachment in both HepG2 and Chinese hamster ovary cells, which were already overexpressing SCCA1. Importantly, in vivo co-expression of human FTL and SCCA1 in mouse liver by means of tailvein hydrodynamic injection increased serum levels of HBV surface antigen transiently 24 hours post challenge with HBV-positive human sera, and a large amount of HBV core antigen-positive hepatocytes around blood vessels could be identified by immunohistochemical staining 48 hours post challenge. The data strongly suggest that FTL and SCCA1 may serve as coreceptors in HBV cellular attachment and virus entry into hepatocytes.

Hydrophobic cavity in C-terminus is essential for hTNF-α trimer conformation.

A variety of tumour necrosis factor α (TNF-α) derivatives have been bioengineered to improve antitumour activity and reduce toxicity. The expression of TNF-α in Escherichia coli usually yields a mixture of homotrimers and monomers; however, only the trimer shows antitumour activity. TNF-αD10, a bioengineered hTNF-α derivative, demonstrated 10-fold higher cytotoxicity against tumour cells compared to hTNF-α, but the trimer to monomer ratio was 58:42. In the present study, we investigated the structural differences between the trimer and the monomer of TNF-αD10. We found that the chemical shifts of the C-terminal Trp(114) in the trimer were significantly different from those in the monomer and that the replacement of Trp(114) with different amino acids remarkably reduced the trimer production. Further analysis of the publicly available X-ray crystallographic data for trimeric and monomeric hTNF-α revealed that the conformation of the U-shaped region formed by the fragment Cys(101)-Trp(114) was different between the two forms: a hydrophilic cavity in the monomer and a hydrophobic cavity in the trimer. These findings suggested the potential approaches of molecular and structural modification for future improvement of hTNF-α trimer production.

The role of VE-cadherin in osteosarcoma cells.

Osteosarcoma cells can generate vasculogenic-like, patterned networks to obtain nutrients and oxygen, which mimic some function of endothelial-like cells and facilitate tumor malignant progress. These cells also express vascular endothelial-cadherin (VE-cadherin), which is generally accepted as a strictly endothelial-specific transmembrane protein. However, its role is still relatively obscure in osteosarcoma cells. So we inhibit the VE-cadherin gene expression with siRNA in osteosarcoma cells (MG63), and culture those cells in three-dimensional medium, containing Type I collagen or Matrigel, to observe the role of VE-cadherin. Western blotting analysis show that sequence-specific siRNA can significantly decrease the expression of VE-cadherin in MG63 cell. After knockdown of VE-cadherin, osteosarcoma cells can't induced angiogenic sprout and form osteosarcoma-generated, endothelial-like networks. Our data indicate that VE-cadherin may be a positive and specific regulator not only in angiogenesis, but also in vasculogenic mimicry of osteosarcoma cells. And it can be considered as a new prospective option in the combining treatment of aggressive tumor with highly vascularity, including osteosarcoma.

Lentivirus-mediated short hairpin RNA targeting the APRIL gene suppresses the growth of pancreatic cancer cells in vitro and in vivo.

RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Herein, we constructed a short hairpin RNA (shRNA) lentiviral expression vector targeting a proliferation-inducing ligand (APRIL) gene in the CFPAC-1 cell (a type of cell strain of human pancreatic cancer) in order to observe the inhibitory effect of APRIL gene's shRNA on the growth of the CFPAC-1 cell in vitro and in vivo. The results showed that lentivirus-mediated RNAi effectively inhibited the expression of APRIL mRNA and protein in CFPAC-1 cells. Moreover, it can inhibit the growth of pancreatic cancer cells in vitro and in vivo. Our study indicates that lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in cancer gene therapy studies.

esiRNA to eri-1 and adar-1 genes improving high doses of c-myc-directed esiRNA effect on mouse melanoma growth inhibition.

Knockdown of c-myc expression via RNAi is expected to be an efficient approach to suppress tumor growth. In our preliminary study, we intraperitoneally injected different doses of c-myc-directed esiRNA (esic-MYC, c-myc-directed Escherichia coli expressed and enzyme digested siRNA) into C57BL6/6J mice with bearing B16 melanoma to investigate the inhibitory effect of esic-MYC on tumor growth. However, in high dose esic-MYC treatment groups, the tumor growth inhibition was less efficient than that of low dose treatment groups. Considering the negative regulation roles of eri-1 and adar-1 genes in RNA interference, we downregulated either/both of the two genes with c-myc gene by RNAi. Our results showed esiMERI-1 (esiRNA of mouse eri-1 gene) and esiMADAR-1 (esiRNA of mouse adar-1 gene) could rescue the tumor growth suppression in the high dose esic-MYC treatment groups obviously. The data strongly suggest that silencing of eri-1 and adar-1 homologs of human being should be concerned for cancer therapy by RNAi approach.

A 14-mer peptide from HSP70 protein is the critical epitope which enhances NK activity against tumor cells in vivo.

Heat shock protein 70 (Hsp70) has been found to play key roles in tumor immunity due its chaperone function of binding antigenic peptides. Here we report it can also stimulate NK cells in vivo, which is another role in Hsp70s' anti-tumor response. Injecting Hsp70 into mice increased splenic NK cell populations, which may be reason for anti-tumor effect of Hsp70. The Hsp70 14-mer peptide (aa450-463, TRD) was identified as the critical epitope for this stimulatory activity. It was the murine Hsp70 14-mer peptide TRD instead of the corresponding human Hsp70 14-mer peptide TKD that functioned in the mouse experimental model.

[Effect of EPHA2-siRNA plasmid on biological behavior of human osteosarcoma cells in vitro].

OBJECTIVE: To investigate the role of EPHA2 in regulating apoptosis, proliferation and vasculogenic mimicry of osteosarcoma cells, by gene silencing through RNA interference. METHODS: EPHA2-siRNA plasmids were achieved by gene cloning. The plasmids were transfected into human osteosarcoma cells (MG63). The expression level of EPHA2 protein was measured by Western blotting. The proliferation, apoptosis and vasculogenic mimicry features of osteosarcoma MG63cells were assessed by light microscopy, MTIP assay, flow cytometry, annexin V-FITC/PI and HE staining, respectively. RESULTS: The EPHA2-siRNA plasmid was confirmed by DNA sequencing. After treatment with Sequence-specific siRNA targeted EPHA2, the protein level of the transfected group decreased significantly. As compared to non-siRNA transfected cells, the transfected group showed lower proliferation, higher and earlier apoptosis and less osteosarcoma-generated vasculogenic mimicry. CONCLUSION: EPHA2 gene may be involoved in apoptosis and proliferation of osteosarcoma cells, and may be necessary for vasculogenic mimicry. Down-regulation of EPHA2 expression by sequence-specific siRNA may be considered as a new option in the treatment of EPHA2 over-expressing cancer including osteosarcoma in future.

Fusion proteins of Hsp70 with tumor-associated antigen acting as a potent tumor vaccine and the C-terminal peptide-binding domain of Hsp70 being essential in inducing antigen-independent anti-tumor response in vivo.

Hsp70s are a family of ATP-dependent chaperones of relative molecular mass around 70 kDa. Immunization of mice with Hsp70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor challenge. In this work, we investigated whether Hsp70 can be used as vehicle to elicit immune response to its covalence-accompanying antigen. A recombinant protein expression vector was constructed that permitted the production of recombinant protein fusing tumor-associated antigen (eg, Mela) to the C terminus of Hsp70. We found that the Hsp70-Mela fusion protein can elicit strong cellular immune responses against murine tumor B16, which expresses protein Mela. The Hsp70 peptide-binding domain deletion mutant of the fusion protein was sufficient for inducing Mela-specific cytotoxic T lymphocyte but was not sufficient for engendering potent anti-tumor immunity against B16. We also found that host natural killer (NK) cells were stimulated in vivo by C-terminal domain of Hsp70. We thus presume that Hsp70 fusion proteins suppress tumor growth via at least 2 distinct pathways: one is covalence-accompanying antigen dependent; another is antigen independent. The C-terminal domain of Hsp70 seemed to be the crucial part in eliciting antigen-independent responses, including NK cell stimulation, against tumor challenges. Furthermore, we found that immunization with multiple Hsp70 fusion proteins resulted in a better anti-tumor effect.

Up-regulation of P-glycoprotein expression by catalase via JNK activation in HepG2 cells.

Overexpression of the MDR1 gene is one of the reasons for multidrug resistance (MDR). Some studies suggested that antioxidants could down-regulate MDR1 expression as a possible cancer treatment. In this report, we try to determine the effects of antioxidants (catalase or N-acetylcysteine [NAC]) on the regulation of intrinsic MDR1 overexpression in HepG2 cells. Adding catalase or N-acetylcysteine to the HepG2 culture led to a significant increase of MDR1 mRNA and P-glycoprotein drug transporter activity. After catalase or NAC treatment, a reduced intracellular reactive oxygen species (ROS) was observed. The JNK inhibitor SP600125 abolished the positive effects of catalase on drug transporter activity in a dose-dependent manner. Furthermore, the up-regulation of P-glycoprotein functions by catalase was only observed in HepG2 cells but not in other cell lines tested (MCF-7, A549, A431). These data suggested that catalase can up-regulate P-glycoprotein expression in HepG2 cells via reducing intracellular ROS, and JNK may mediate this process.

Blocking c-myc and stat3 by E. coli expressed and enzyme digested siRNA in mouse melanoma.

Tumour cells often show alteration in the signal-transduction pathways, leading to proliferation in response to external signals. Oncogene overexpression and constitutive expression is a common phenomenon in the development and progression of many human cancers. Therefore oncogenes provide potential targets for cancer therapy. RNA interference (RNAi), mediated by small interfering RNA (siRNA), silences genes with a high degree of specificity and potentially represents a general approach for molecularly targeted anti-cancer therapy. The data presented in this report evaluated the method of systemically administering combined esiRNAs to multiple targets as compared with the method of using a single kind of esiRNA to a single target. Our experimental data revealed that the mixed treatment of esiC-MYC and esiSTAT3 had a better inhibition effect than the single treatment of esiC-MYC or esiSTAT3 on mouse B16 melanoma.

Fusion protein of ATPase domain of Hsc70 with TRP2 acting as a tumor vaccine against B16 melanoma.

HSP70s are a family of ATP-dependent chaperones of relative molecular masses around 70kDa. Immunization of mice with HSP70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor. Recent researches have demonstrated that the ATPase domain of HSP70 and the tumor antigenic peptide that binds to Hsp70 were the crucial parts eliciting tumor-specific immunity. These findings suggested that a recombinant protein expressed in Escherichia coli, comprising a covalently fused fragment of tumor rejection antigen to ATPase domain of HSP70, could be used as a tumor vaccine. However, high-level expressions of heterologous recombinant proteins in E. coli often lead to the formation of inclusion bodies, resulting in defects in solubility and bioactivity. In the present work, we found an approach to resolve these problems, focusing on a refolding procedure via gel-filtration chromatography for denatured inclusion body proteins. Here, we expressed, purified and refolded a fusion protein comprising murine heat shock cognate protein 70 (Hsc70) N-terminal ATPase domain (Hsc70NTD) and a portion of TRP2 (aa153-417) as a model protein. The refolding effectivities were assessed according to their ATPase activities, the vaccine function was assessed according to immunization effect in inducing antigen-specific CTLs and to in vivo tumor protection. The results showed that the fusion protein refolded via gel-filtration chromatography exhibited ATPase activity, succeeded in eliciting antigen-specific CTL in vivo and delayed tumor growth on tumor-bearing mice.

Reversal of MDR1 gene-dependent multidrug resistance using low concentration of endonuclease-prepared small interference RNA.

Multidrug resistance following initial chemotherapy is commonly associated with MDR1 gene encoding for P-glycoprotein (P-gp). RNA interference of MDR1 gene expression was used as a strategy to reverse MDR1-mediated multidrug resistance phenotypes. Here we report that endonuclease-prepared small interfering RNA (esiRNA) at concentrations as low as 10 ng/ml (about 0.7 nM) can decrease MDR1 expression and increase chemosensitivity in the Adriamycin-induced resistant MCF-7/R cells. When MCF-7/R cells were transiently transfected with esiRNA of MDR1 (esiMDR1), the MDR1 mRNA was reduced by about 50%, drug accumulation increased by about 30%, and the IC50 for daunorubicin was reduced from 4.5 to 1.2 microM. These results provide evidence that esiRNA of MDR1 could be an alternative to P-gp inhibitors with the advantage of avoiding non-specific suppression with a lower effective dosage than using a single siRNA duplex, offering a potential therapeutic application of siRNA.

esiRNAs purified with chromatography suppress homologous gene expression with high efficiency and specificity.

Many preclinical studies have shown RNA interference (RNAi) as a new promising way to treat various human diseases including cancer and virus infection and there is an increasing demand for the large-scale preparation of short interfering RNAs (siRNAs) at low cost. Data are accumulating to show that endoribonuclease-prepared siRNAs (esiRNAs) are superior to chemically synthesized siRNAs in terms of expense, efficiency, and specificity. Yet all procedures available for esiRNA purification were designed to produce small amount of siRNAs for laboratory use. In this article, a new method of purification of esiRNAs based on ion exchange chromatography and size exclusion chromatography is reported. The esiRNAs prepared with this method are shown here to be of high purity and specifically suppress homologous gene expression without activating interferon response and with higher efficiency than chemically synthesized siRNAs. We can expect that the new method can be scaled up easily to provide large quantities of esiRNAs to meet the requirement of preclinical and clinical studies.

Comparison of the anti-tumor effects of various whole-body hyperthermia protocols: correlation with HSP 70 expression and composition of splenic lymphocytes.

Whole-body hyperthermia (WBH) has been used as an adjunct approach to radio-/ chemotherapy for tumor therapy for many years. However, the molecular mechanism underlying the enhancement of tumor control is not clearly understood. It has been hypothesized that WBH might activate immune system by inducing the expression of heat shock proteins (HSPs), which are thought to facilitate the presentation of tumor-specific antigens. In the present work, we examined the effects of various thermal doses of WBH on tumor growth delay and HSP70 levels in tumors on C57BL/6 mice, as well as on splenic lymphocyte subpopulations. The maximal WBH effect (about 40% decrease in tumor weight) was achieved by a 2-hour WBH treatment everyday at 40.0 degrees C. By using this treatment schedule, the populations of CD3+/CD4+ T cells and CD3+/CD8+ T cells increased by 4 and 3 times, respectively, at the end of WBH treatment period. When the length of day-by-day WBH treatment was longer than 2 hours or the frequency of WBH treatment was lower than once a day, the effect of tumor growth delay and the population of CD3+ T lymphocyte in spleen increase were discounted. On the other hand, the HSP70 levels in tumor nodules rose continuously as the WBH treating time increased, but the populations of NK cells in spleen did not change significantly. The results suggest that an increased CD3+ T lymphocyte population is closely related to the anti-tumor effect of WBH, which might be a useful marker for effectiveness of hyperthermia. However, neither the levels of HSP70 nor the NK cell populations in spleen appear to correlate to tumor control.

High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse.

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.

Cost-effective method of siRNA preparation and its application to inhibit hepatitis B virus replication in HepG2 cells.

AIM: To find a cost-effective method of preparation of short interfering RNAs based on cloning, fermentation, digestion and purification (CFDP) and test its feasibility to inhibit hepatitis B virus replication in cell culture. METHODS: We constructed an expression vector containing T7 and tac promoter in a head-to-head orientation. cDNA fragment of interest was cloned into this vector between the opposing promoters. dsRNAs were expressed with this vector in Escherichia coli, and purified by affinity chromatography using CF 11 column. They were digested by RNase III in a buffer containing manganese ions, then separated on 15% non-denaturing PAGE, and the siRNAs about 25 bp in length were recovered. siRNAs prepared with CFDP were co-transfected with target gene expression plasmid into human cell lines with lipofectamine 2,000 to test their inhibition efficiency. RESULTS: siRNAs corresponding to part of the hepatitis B virus polymerase gene (siHBVP) prepared by CFDP specifically and dramatically suppressed the virus protein expression. The HBsAg expression level was reduced to 10% that of the control by co-transfection of 60 nmol/L siHBVP in SMMC7721 cells. Dose-dependent effect on suppression of HBsAg and HBeAg expression was observed in HepG2 cells. The highest inhibition rate was kept at 70% during the six days after transfection of 7.5 nmol/L siHBVP. CONCLUSION: We show CFDP is a very promising method to prepare therapeutic agents in anti-virus applications.

Reduction of PTP1B by RNAi upregulates the activity of insulin controlled fatty acid synthase promoter.

Metabolic deregulation accompanying type II diabetes is characterized by insulin resistance in peripheral tissues (liver, muscle, and adipose), mediated by impairments in insulin receptor (IR) signaling. Protein tyrosine phosphatase 1B (PTP1B) has been shown to be a negative regulator of IR autophosphorylation and thus has been considered as a major therapeutic target for the treatment of type II diabetes. We use RNA interference technique to downregulate PTP1B expression in hepatoma cell line. A secretory HBV s-antigen was introduced as reporter and driven by mouse fatty acid synthase promoter, which is positively controlled by insulin signaling. Liver-targeted hydrodynamic injection in tail vein was introduced to transfer siRNA (or siRNA expression vector) and reporter plasmid into mouse liver. On fasted/refed and glucose stimulation condition, the HBV s-antigen in sera in RNAi group was higher than that in the negative group. Our results provided evidence that upregulation of insulin signaling by reducing PTP1B liver with RNAi can be a potent diabetes treatment method.