NaHCO3 enhances the antitumor activities of cytokine-induced killer cells against hepatocellular carcinoma HepG2 cells.

The extracellular pH is lower inside solid tumors than in normal tissue. The acidic environment inhibits the cytotoxicity of lymphocytes in vitro and promotes tumor cell invasion. In the present study, both in vitro and in vivo experiments were conducted to investigate how NaHCO3 would affect the antitumor activities of cytokine-induced killer (CIK) cells against hepatocellular carcinoma (HCC) cells. For the in vitro experiments, HepG2 cells were cultured at pH 6.5 and 7.4 in the presence of CIK cells or CIK cell-conditioned medium (CMCIK). For the in vivo experiments, nude mice were xenografted with HepG2-luc cells and divided into four groups: i) CIK cells injection plus NaHCO3 feeding; ii) CIK cells injection plus drinking water feeding; iii) normal saline injection plus NaHCO3 feeding; and iv) normal saline injection plus drinking water feeding. The results indicated that the viability and growth rate of HepG2 cells were remarkably suppressed when co-cultured with CIK cells or CMCIK at pH 7.4 compared with those of HepG2 cells cultured under the same conditions but at pH 6.5. In the xenograft study, a marked synergistic antitumor effect of the combined therapy was observed. NaHCO3 feeding augmented the infiltration of cluster of differentiation 3-positive T lymphocytes into the tumor mass. Taken together, these data strongly suggest that the antitumor activities of CIK cells against HepG2 cells were negatively affected by the acidic environment inside the tumors, and neutralizing the pH (for example, via NaHCO3 administration), could therefore reduce or eliminate this influence. In addition, it should be recommended that oncologists routinely prescribe soda water to their patients, particularly during CIK cell therapy.

The Tumor-promoting Effects of FAM92A1-289 in Cervical Carcinoma Cells.

BACKGROUND/AIM: FAM92A1-289 is recognized as one of the newly-discovered putative oncogenes. This study was performed to reveal its oncogenic functions in human cervical carcinoma cells. MATERIALS AND METHODS: The FAM92A1-289+ cell line was established with knock-in technique and selected by puromycin-resistance screening. Scratch assay, methylthiazol tetrazolium assay, colony forming assay and xenograft test were used to examine cell migration, cell proliferation, cell viability and tumor formation, respectively. RESULTS: FAM92A1-289+ cells showed higher migration rate (p<0.05), higher cell viability (p<0.01), higher colony formation and tumor growth. The FAM92A1-289 protein was pulled-down by antibodies against proliferating cell nuclear antigen (PCNA) in the co-immunoprecipitation assay. CONCLUSION: The up-regulated expression of FAM92A1-289 could facilitate cell migration, boost cell proliferation and promote colony formation in vitro and tumor growth in vivo. The interaction between FAM92A1-289 and PCNA was verified by co-immunoprecipitation. This study provided functional evidence for FAM92A1-289 to be developed as a therapeutic target for cancer treatment.

The application of mRNA-based gene transfer in mesenchymal stem cell-mediated cytotoxicity of glioma cells.

Since the tumor-oriented homing capacity of mesenchymal stem cells (MSCs) was discovered, MSCs have attracted great interest in the research field of cancer therapy mainly focused on their use as carries for anticancer agents. Differing from DNA-based vectors, the use of mRNA-based antituor gene delivery benefits from readily transfection and mutagenesis-free. However, it is essential to verify if mRNA transfection interferes with MSCs' tropism and their antitumor properties. TRAIL- and PTEN-mRNAs were synthesized and studied in an in vitro model of MSC-mediated indirect co-culture with DBTRG human glioma cells. The expression of TRAIL and PTEN in transfected MSCs was verified by immunoblotting analysis, and the migration ability of MSCs after anticancer gene transfection was demonstrated using transwell co-cultures. The viability of DBTRG cells was determined with bioluminescence, live/dead staining and real time cell analyzer. An in vivo model of DBTRG cell-derived xenografted tumors was used to verify the antitumor effects of TRAIL- and PTEN-engineered MSCs. With regard to the effect of mRNA transfection on MSCs' migration toward glioma cells, an enhanced migration rate was observed with MSCs transfected with all tested mRNAs compared to non-transfected MSCs (p<0.05). TRAIL- and PTEN-mRNA-induced cytotoxicity of DBTRG glioma cells was proportionally correlated with the ratio of conditioned medium from transfected MSCs. A synergistic action of TRAIL and PTEN was demonstrated in the current co-culture model. The immunoblotting analysis revealed the apoptotic nature of the cells death in the present study. The growth of the xenografted tumors was significantly inhibited by the application of MSCPTEN or MSCTRAIL/PTEN on day 14 and MSCTRAIL on day 28 (p<0.05). The results suggested that anticancer gene-bearing mRNAs synthesized in vitro are capable of being applied for MSC-mediated anticancer modality. This study provides an experimental base for further clinical anticancer studies using synthesized mRNAs.

TRAIL-engineered bone marrow-derived mesenchymal stem cells: TRAIL expression and cytotoxic effects on C6 glioma cells.

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL) is considered as a tumor cell-specific cytotoxic agent. Through the aid of mesenchymal stem cells (MSCs), TRAIL is capable of inducing apoptosis of tumor cells in tumor sites. The present study was performed to investigate the cytotoxic effects of TRAIL-engineered MSCs on glioblastoma cells (C6) in vitro. MATERIALS AND METHODS: An expression vector of secreting form of TRAIL was used to engineer MSCs. The cytotoxic effects of TRAIL-transfected MSCs on C6 cells were invstigated using the MTT method and Hochest33258 staining after co-culture of the two cell types. RESULTS: TRAIL and control plasmid transfection of MSCs showed no significant effect on MSC's viability (p>0.05). A significant inhibition of C6 cells was observed when they were co-cultured with TRAIL-engineered MSCs (63.7%±0.12, p<0.05). CONCLUSION: Mesenchymal stem cells were very well tolerant to the transfection of TRAIL-bearing vectors. The cytotoxic effects of TRAIL-engineered MSCs on C6 cells indicates the therapeutic potential of this strategy for treatment of glioblastoma patients.

Cryosurgery and rhTNF-α play synergistic effects on a rat cortex C6 glioma model.

Glioma, a type of brain tumor originating from glioma cells, varies widely in aggressiveness and causes serious symptoms, but the treatments are limited. Studies have shown that cryosurgery has multiple effects on tumor treatments, and administration of human tumor necrosis factor-alpha (rhTNF-α) arguments the anti-tumor effect of cryotherapy in breast and prostate cancers. To test the hypothesis that cryosurgery and rhTNF-α play synergistic effects against brain tumors, we established a brain glioma model on rat cortex regions following different treatments: the G1 group was sham-operated; the G2 group was treated with cryosurgery; the G3 group was treated with rhTNF-α; and G4 group received combined treatment with cryosurgery and rhTNF-α. Tumor sizes were measured by magnetic resonance imaging; DNA fragmentation was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay); P21(WAF1/CIP1) and proliferating cell nuclear antigen (PCNA) expression levels were scored using immunohistochemical staining. G2 and G4 rats had significantly longer survival time than did G1 rats. Tumor sizes in each group were significantly decreased as compared with those in G1 rats. PCNA-positive cells were significantly decreased in G2, G3 and G4 rats as compared with G1 rats. In contrast, DNA fragmentation and P21(WAF1/CIP1)-positive cells were significantly increased in each treatment group. Importantly, a combined treatment enhanced the effects of cryosurgery. Combined treatment with cryosurgery and rhTNF-α may have a synergistic effect on glioma tumor therapy, enhancing the inhibition of proliferation and the induction of apoptosis.

A protocol for islet isolation from mouse pancreas.

Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. We describe a protocol for islet isolation from mouse pancreas. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30-45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.

Do immunotherapy and beta cell replacement play a synergistic role in the treatment of type 1 diabetes?

Type 1 diabetes (T1D) is the result of the autoimmune response against pancreatic insulin-producing ss-cells. Its ultimate consequence is beta-cell insufficiency-mediated dysregulation of blood glucose control. In terms of T1D treatment, immunotherapy addresses the cause of T1D, mainly through re-setting the balance between autoimmunity and regulatory mechanisms. Regulatory T cells play an important role in this immune intervention. An alternative T1D treatment is beta-cell replacement, which can reverse the consequence of the disease by replacing destroyed beta-cells in the diabetic pancreas. The applicable insulin-producing cells can be directly obtained from islet transplantation or generated from other cell sources such as autologous adult stem cells, embryonic stem cells, and induced pluripotent stem cells. In this review, we summarize the recent research progress and analyze the possible advantages and disadvantages of these two therapeutic options especially focusing on the potential synergistic effect on T1D treatment. Exploring the optimal combination of immunotherapy and beta-cell replacement will pave the way to the most effective cure for this devastating disease.

[Effect of cryotherapy and 5-fluorouracil on apoptosis of G422 glioma cells].

BACKGROUND & OBJECTIVE: It was unknown whether the combination of cryotherapy and 5-fluorouracil (5-FU) could produce a synergistic effect, though both of them could induce apoptosis of glioma cells. The aim of this study was to observe the effect of cryotherapy and 5-fluorouracil on apoptosis of G422 glioma cells and to investigate the potential mechanism. METHODS: (1)Experimental mice models with G422 glioma were established. Cryotherapy (-180 degrees C,90s),chemotherapy (5-FU, 18 mg/kg), and cryochemotherapy were performed on them,respectively.(2)TUNEL was used to determine the apoptosis of glioma cells in each group at different time points.(3)The expression of the p53 and the HSP90alpha gene of the frozen glioma were analyzed by immunohistochemical staining. RESULTS: Cryotherapy or 5-FU induced apoptosis of G422 glioma cells, while the combination of cryotherapy and 5-FU remarkably increased the apoptosis rate. The numbers of apoptotic cells of combination group were 30.6+/-11.7, 86.4+/-21.5, 128.1+/-4.1, 237.0+/-30.1, 72.8+/-23.0 at 6h, 12h, 24h, 48h, 72h after cryochemotherapy, respectively, which were significantly higher than those of other groups. There was no expression of HSP90alpha and p53 in the center of freezing area. The p53 and the HSP90alpha proteins increased obviously in the periphery area especially at 48h after freezing compared with control group [(73.1+/-9.3)% vs (60.6+/-9.9)%, (35.6+/-6.6)% vs (13.7+/-6.5)%](P< 0.01). The expression of p53 was positively correlated with expression of HSP90alpha in frozen glioma (r=0.3610, P< 0.01). CONCLUSION: Combination of cryotherapy with 5-FU could enhance the apoptosis of G422 glioma cells presumably through modulating the HSP90alpha and p53 expression pattern. Cryochemotherapy showed better effect than that of cryotherapy or chemotherapy alone.