Corrigendum to "Suppression of IL-6 Gene by shRNA Augments Gemcitabine Chemosensitization in Pancreatic Adenocarcinoma Cells".

[This corrects the article DOI: 10.1155/2018/3195025.].

Promoting the formation of Pi-stacking interaction to improve CTL cells activation between modified peptide and HLA.

OBJECTIVE: This study aims to investigate the use of single residue substitution to promote the formation of pi-stacking interactions between peptides and Human leukocyte antigen (HLA)-A*2402 molecules to improve the affinity of peptides and HLA molecules, as well as the level of cytotoxic T lymphocyte (CTL) cells activated by peptides-HLA (p-HLA) complex. METHODS: Molecular docking and molecular dynamics simulation were used to simulate and analyze the interactions and binding free energies between HLA-A*2402-restricted antigen peptides and HLA molecules, before and after the single residue substitution. HLA-A*2402 restricted antigen peptides before and after the single residue replacement were loaded into dendritic cells (DCs) in vitro, and further Enzyme-Linked ImmunoSpot (ELispot) test was carried out to evaluate the effect of modified antigen peptides on the immune activation of CTL cells. RESULT: After replacing the antigen peptides with a single residue, some of them could promote the formation of pi-stacking interaction. The binding free energy between the modified antigen peptides and HLA-A*2402, as well as the level of immune activation of CTL cells were mostly higher than before, especially after the replacement of the 9th residue of the polypeptide, such as C9F and C9W. There was a significant negative correlation between the level of activated CTL cells by modified antigen peptides and the total interaction amount of hydrogen bonds and salt bridges. CONCLUSION: Promoting the formation of pi-stacking interaction between antigen peptides and HLA-A*2402 molecules could increase the total binding free energy of p-HLA complex and the level of CTL cells activation. In addition, the amount of hydrogen bonds and salt bridges between peptides and HLA could reduce the level of immune activation. All the characteristics above can improve the immunogenicities of the weak antigens.

Suppression of IL-6 Gene by shRNA Augments Gemcitabine Chemosensitization in Pancreatic Adenocarcinoma Cells.

Pancreatic adenocarcinoma has an exceedingly poor prognosis, accounting for five-year survival of less than 5%. Presently, improving the efficacy of pancreatic adenocarcinoma treatment has been the focus of medical researchers worldwide. Recently, it has been suggested that deregulation of interleukin- (IL-) 6 is caused by a key gene involved in the beginning and development of pancreatic adenocarcinoma. Herein, we investigated whether suppression of IL-6 could augment gemcitabine sensitivity in the PANC-1 cells. We found considerably higher expression of IL-6 in pancreatic adenocarcinoma tissues than that in the adjacent nontumorous tissues. Suppression of IL-6 by shRNA resulted in apoptosis as well as inhibition of cell proliferation and tumorigenicity. In addition, suppression of IL-6 remarkably promoted antitumor effect of gemcitabine, indicating that the combination of shRNA targeting IL-6 with gemcitabine may provide a potential clinical approach for pancreatic cancer therapy.

Radiosensitization effect of hsa-miR-138-2-3p on human laryngeal cancer stem cells.

BACKGROUND: Treatments that target cancer stem cells play an important role in the controlling and eliminating of tumor initiation as well as in development, progression, and chemotherapy/radiotherapy resistance. In our previous study, we cultured and harvested human laryngeal cancer stem cells (CSCs) and applied microRNA biochips to screen differentially expressed miRNAs that were related to radiation tolerance in irradiated human laryngeal CSCs. According to the predicted genes and pathways of differential miRNAs target, down-regulated expression of hsa-miR-138-2-3p under radiation was thought to play a key role in enhancing the radio-sensitivity in human laryngeal squamous cancer stem cells. METHOD: To investigate the radiational enhancement of hsa-miR-138-2-3p, we transfected hsa-miR-138-2-3p mimics that were synthesized based on the sequences of hsa-miR-138-2-3p in vitrointo human laryngeal CSCs (Hep-2, M2e, and TU212 cell lines) to make hsa-miR-138-2-3p overexpressed, and the tumorous specialities of CSCs, like cell proliferation, invasion, apoptosis, cell cycle arrest, and DNA damage were evaluated by CCK-8 assay, clone formation assay, invasion assay, flow cytometry, and comet assay. Furthermore, we explored the signal transduction pathways that regulated the cancer stem cell initiation, development, invasion, apoptosis and cell cycle arrest, which were controlled by hsa-miR-138-2-3p. RESULT: Overexpressed hsa-miR-138-2-3p played a key role in many anti-cancer biological processes in human laryngeal CSCs: (1) it decreased laryngeal CSCs proliferation and invasion in response to radiotherapy; (2) it increased the proportion of early and late apoptosis in laryngeal CSCs after radiation, raised G1 phase arrest in laryngeal CSCs after radiation, and decreased the proportion of S stage cells of cell cycle that were related to radio-resistance in laryngeal CSCs; (3) it down-regulated the expression of ß-catenin in Wnt signal pathway that was related to the tolerance of laryngeal CSCs to radiotherapy; (4) it down-regulated the expression of YAP1 in Hippo signal pathway that regulated cell proliferation, invasion and apoptosis; (5) it up-regulated the expression of p38 and JNK1 in MAPK signal pathway that was concerned to radio-sensitivity. CONCLUSION: In the present study, it was found that hsa-miR-138-2-3p regulated the Wnt/ß-catenin pathways, the Hippo/YAP1 pathways, and the MAPK/p38/JNK1 pathways that were involved in cell proliferation, invasion, apoptosis, cell cycle arrest, radio-resistance and radio-sensitivity in laryngeal CSCs. These results will be useful for a better understanding of the cell biology of hsa-miR-138-2-3p in laryngeal CSCs, and for serving hsa-miR-138-2-3p as a promising biomarker and as a target for diagnosis and for novel anti-cancer therapies for laryngeal cancers.

Screening for MiRNAs related to laryngeal squamous carcinoma stem cell radiation.

OBJECTIVE: To use microarray chip technology for screening of stem cell radiation related miRNAs in laryngeal squamous cell carcinoma; study and explore the relationship of miRNAs with radiosensitivity of laryngeal squamous cells. METHOD: After conventional culture and amplification of the laryngeal squamous carcinoma cell line Hep-2, CD 133+ cells were screened out with combination of isolated culture of stem cell microspheres and FACS for preparation of laryngeal cancer stem cells. After radiation treatment, miRNAs of laryngeal squamous carcinoma stem cells before and after radiation were enriched and purified. After microarray hybridization with mammalian miRNA and scanning of fluorescence signal, the miRNAs of laryngeal squamous carcinoma stem cells before and after radiation was subject to differential screening and clustering analysis. Real-time quantitative RT-PCR was used to verify part of the differentially expressed miRNAs. RESULTS: 70 miRNAs related to laryngeal cancer stem cell radiation with 2-fold difference in expression were screened out, in which 62 were down-regulated and 8 were up-regulated. Fluorescent quantitative RT-PCR results were consistent with miRNAs chip results. CONCLUSION: Some miRNAs may be involved in self-regulation with laryngeal squamous carcinoma stem cell radiation.

[Influence of FOLFOX regimen on the immunologic function in patients with advanced colorectal cancer].

OBJECTIVE: To examine the influence of chemotherapy with FOLFOX protocol (CT-F) on the immunologic function in patients with advanced colorectal cancer. METHODS: A total of 43 patients with advanced colorectal cancer were included. Patients who received FOLFOX chemotherapy (Group A, n=22) were compared to those who did not(Group B, n=21). Blood was obtained from peripheral vein before chemotherapy (T0), at the time of completion of chemotherapy (T1), and 3 months after completion of chemotherapy (T2) to detect the percentage of regulatory T lymphocytes (CD4+CD25+Foxp3+T cells) in total T lymphocytes using fluorescence activated cell sorter (FACS). The level of Th1/Th2 cytokines in the serum including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-γ (IFN-γ) were detected by enzyme-linked immunoabsorbent assay(ELISA). RESULTS: The percentage of regulative T lymphocyte was significantly lower at T1, which increased after chemotherapy but was still lower than that at T0 and that in Group B [(4.15±0.56)%, (5.60±0.88)%, and(5.38±0.92)%, all P<0.01]. The levels of IL-4, IL-10 decreased at T1, and increased to the normal level at T2 compared with those at T0 or those in the control group (all P<0.01). In contrast, the levels of IL-2 and IFN-γ increased significantly at T1 and decreased to the normal level at T2 during the entire observation period. CONCLUSION: For patients with advanced colorectal cancer, FOLFOX chemotherapy can decrease the proportion of regulatory T lymphocytes and results in Th1/Th2 cytokine drift to Th1 type. Therefore, FOLFOX may help the release from the anticancer immune inhibition.

[Preparation of anticolon carcinoma vaccine with rich chaperone peptides and study on its anticancer efficacy].

OBJECTIVE: To prepare the anticolon carcinoma vaccine with rich chaperone peptide and to examine its anticancer immunological efficacy. METHODS: CT26 colon carcinoma cells were cultured in 1 mg/L Trichosanthin 1640 medium at different temperatures to induce the chaperone expression and promote the synthesis of antigen peptides. Groups of these cells treated under the different condition were lysed by the sonic disintegration, and the lysates were centrifuged. The rawpurified proteins were obtained from the supernatants by precipitating with saturated ammonium sulfate and removing the molecules below 50,000 and above 300,000 in molecular weight via dialysis. Furthermore, the proteins with the molecular weights in 70,000, 90,000, 95,000, 110,000 and 170,000 were collected through gel filtration and SDS-PAGE. The purified proteins were analysed by Western blotting, and inspected on the anticancer immunological effects including lymphocyte proliferation and the activities of NK and CTL. RESULTS: Major of the chaperone peptides of anticancer effects in CT26 cells, including antigen peptides joining with HSP70, HSP90, gp96, HSP110 and HSP170, was satisfactorily extracted and condensed, and rich chaperone peptide composites were successfully obtained. The composites prepared under various condition could all enhance lymphocyte proliferation and the activities of CTL and NK(P<0.01). CONCLUSIONS: The rich chaperone peptide composites are successfully prepared via dialysis, salt fractionation and gel filtration combined with SDS-PAGE. Both the heat stress and Trichosanthin can increase the composites, which treated by 42 centi-degree heat stress and Trichosanthin are found to possess the strongest anticancer efficacy.

[Influence of chemotherapy with FOLFOX protocol on sex hormones of male patients and the protective effect of herbal medicines for reinforcing Shen and supplementing qi on it].

OBJECTIVE: To observe the influence of chemotherapy with FOLFOX protocol (CT-F) on sex hormones of male patients, and the protective and detoxifying effect of herbal medicines for reinforcing Shen and supplementing qi (HM) on it. METHODS: The randomized block control and self-control design was adopted to retrospectively investigate the changes of sex hormones in 61 patients with cancer of colon, rectum or stomach. They were assigned to four groups, A: treated simply with HM; B: treated with CT-F; C: treated with CT-F combined with HM; D: the blank control group. One course of CT-F composed of 1 month treatment, and 6 courses totally were applied on each patients. The levels of luteinizing hormone (LH), estradiol (E2), prolactin (PRL), progesterone (P), testosterone (T) and follicle-stimulating hormone (FSH) were determined before treatment (T0), at the end of the 6th month treatment (T1) and the 12th month (T2) after starting treatment. RESULTS: Levels of LH, PRL and T were significantly lowered in the group B at T1, being 4.6 +/- 0.4 IU/L, 8.6 +/- 0.7 microg/L and 13.1 +/- 1.4 IU/L, respectively, which were significantly different to those in the other 3 groups at the corresponding time; they were somewhat raised after chemotherapy but still lower at T2 than those at T0, being 5.0 +/- 0.4 IU/L, 9.9 +/- 1.1 microg/L and 14.1 +/- 1.4 IU/L respectively, also lower than the corresponding levels in the other 3 Groups (P<0.05 or P<0.01). In group C, LH significantly lowered at T1 (P<0.05) to 5.1 +/- 0.4 IU/L, but it was restored to the levels of T0 and that in Group D, reaching 6.1 +/- 0.5 IU/L; while PRL and T were changed insignificantly in the chemotherapeutic course, and restored at T2 to the level of T0, comparable to that in group D. Contrarily, levels of E2 and FSH increased significantly (P <0.01) in group B after chemotherapy (at T1) to 135 +/- 14 pmol/L and 9.1 +/- 1.1 IU/L respectively, and till T2, being 140 +/- 15 pmol/L and 9.1 +/- 1.o IU/L, they were lower than those at T0 and higher than those in group D, A and C ( all P <0.01), but the two indexes were not significantly increased in group C, being 116 +/- 12 pmol/L and 7.1 +/- 0.9 IU/L at T1. However, all the parameters showed no significant change in group A and D, and the level of P showed insignificant change in all the groups in the whole observation period. CONCLUSION: CT-F could induce multiple sex hormonal abnormalities in male patients with post-operational gastrointestinal cancer, and HM shows protective and detoxifying effects on them in different degrees.

[Antitumour effect of total saponins of Rubus parvifolius on malignant melanoma].

OBJECTIVE: To evaluate the antitumor effect of total saponins of R. parvifolius on malignant melanoma. METHOD: The human malignant melanoma A375 cells were regularlly subcultured in vitro, and were divided into five groups contained positive control group (CTX), high concentration (0.01 mg x mL(-1)) and middle concentration (0.001 mg x mL(-1)) and low concentration (0.000 1 mg x mL(-1)) total saponins of R. parvifolius groups and negative control group. By using MTT colorimetric method, the cell viability was measured. B16 melanoma cells were transplanted to mice, which were divided into positive control group, high dose (100 mg x kg(-1)) and middle dose (50 mg x kg(-1)) and low dose (25 mg x kg(-1)) total saponins of R. parvifolius groups and negative control group. The inhibition effect of the tumor in vivo, mean survival time and rate of life-elongation of the mice were observed. TUNEL method was used to detect the apoptosis of B16 malignant melanoma. RESULT: Antitumor assay in vitro showed that the absorbency increased in the concentration of 0.01, 0.001 mg x mL(-1) with statistical significance (P < 0.05 vs negative control). Antitumor assay in vivo showed that the tumor inhibitory rate of high dose (100 mg x kg(-1)) and middle dose (50 mg x kg(-1)) of total saponins of R. parvifolius were 37.02% and 30.61%, respectively. Loaded tumor mouse survival duration could be prolonged. The apoptosis indexes of B16 tumor cells in three treatment groups were 32.5%, 20.5% and 5.5%, respective and there was statistical significance (P < 0.05 vs negative control). CONCLUSION: The total saponins of R. parvifolius has remarkable inhibition of proliferation of malignant melanoma cells in vivo and in vitro and exerts antitumor activities through promoting tumor cell apoptosis.

[Anticancer therapeutic effect of SEA-linked and membrane-bound HSP70-expressed intestine-carcinoma vaccine].

OBJECTIVE: To develop a novel dual-modified vaccine, the superantigen-linked intestine-carcinoma cells expressing membrane-bound heat shock protein 70 (HSP70), and further examine its anticancer therapeutic effect. METHODS: The pre-established intestine carcinoma CT26 line expressing membrane-bound heat shock protein 70 (HSP70) was amplified and incubated with superantigen fusion protein, staphylococcal enterotoxin A (SEA) fused with transmembrane sequence (SEA-TM), thereby the dual-modified vaccine was prepared after inactivation. The anticancer efficacy of the vaccine was examined. RESULTS: The laser confocal microscopy and flow cytometry showed that there co-existed much HSP70 and SEA on the vaccine membrane surface. Both of the single-modified vaccines, the SEA-linked vaccine and membrane-bound-HSP70-expressing one, displayed marked tumor suppression, a prolonged survival period, augmented lymphocyte proliferation and higher NK and CTL activity in the vaccinated mice when compared with its counterpart. Furthermore, the dually modified vaccine induced lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest tumor rejection in the vaccinated mice. The survival period of the mice was further prolonged. CONCLUSION: A new vaccine, SEA-linked and membrane-bound-HSP70-expressing intestine-carcinoma cells can induce more potent anticancer immunity and produce better therapeutic efficacy.

[Antitumor therapeutic effect induced by intestine-carcinoma cells vaccine expressing membrane-bound heat shock protein 70].

OBJECTIVE: To develop a new vaccine expressing membrane-bound heat shock protein 70 (mbHSP70) and further study its antitumor therapeutic effect. METHOD: The pre- established vector expressing mbHSP70 was transfected into CT26 cells of colorectal cancer. After the CT26 cells were incubated with 900 microg/ml G418, the sub-clones resistant to G418 were harvested and the HSP70 positive clones were selected by limiting dilution. The clones were amplified and inactivated, thereby the vaccine expressing mbHSP70 was prepared. Lymphocyte proliferation stimulated by the vaccines, NK and CTL activity was observed. The antitumor efficacy of vaccine was observed in BALB/c mice model with colorectal cancer. RESULTS: The laser confocal microscopy and flow cytometry showed that there existed much HSP70 on the vaccine membrane surface. The HSP70 gene-modified vaccine displayed augmented lymphocyte proliferation and higher NK and CTL activity in vitro,and marked tumor suppression and prolonged survival time of the vaccinated micein vivo, when compared with its counterpart. Furthermore, mbHSP70-expression vaccine elicited lymphocyte proliferation most intensively, generated the highest NK and CTL activity as well as the strongest antitumor effect, and prolonged survival time of the vaccinated mice. CONCLUSION: A new vaccine expressing mbHSP70 has more potent antitumor immunity and better therapeutic efficacy than HSP70 gene-modified vaccine did.

[Preparation of mB7.1-GPI and SEA-TM dual-anchored tumor cell vaccine and its antitumor effect].

OBJECTIVE: To prepare the SEA-TM and mB7.1-GPI dual-anchored EL-4 cell vaccine and to investigate its antitumor effects. METHODS: mB7.1-GPI-anchored EL-4 cell vaccine, SEA-TM-anchored EL-4 cell vaccine, SEA-TM and mB7.1-GPI dual-anchored EL-4 cell vaccine were prepared. In vitro the biological activities of these vaccines were measured using a lymphocyte proliferation assay and cytokine release assay on splenocytes derived from C57BL/6 mice. The splenocytes were co-cultured with EL-4 or EL-4/mB7.1-GPI or EL-4/SEA-TM or EL-4/SEA-TM + mB7.1-GPI (treated with Mitomycin C). Lymphocyte proliferation was determined with MTT assay, the concentrations of cytokines (IL-2 and IFN-gamma) were measured using a ELISA technique. Forty C57BL/6 mice were inoculated with EL-4 cells, after 3 days the mice were randomly divided into 5 groups with 8 in each and were treated with PBS, EL-4 cell vaccine, EL-4/mB7.1-GPI cell vaccine, EL-4/SEA-TM cell vaccine and EL-4/SEA-TM + mB7.1-GPI cell vaccine respectively, vaccines were injected three time with two-day interval. Animals were observed daily, tumor sizes were measured every third day. Twenty-five days after tumor challenge, 3 mice in each group were sacrificed and splenic lymphocytes were isolated to examine the activity of natural killer cells (NK) and cytolytic T lymphocytes (CTL). The survival of the remaining 5 mice in each group was observed till the 90th day. RESULTS: mB7.1-GPI or/and TM-SEA fusion protein was stably anchored onto the surface of EL-4 tumor cells. EL-4/mB7.1-GPI or EL-4/SEA-TM had a stronger ability to stimulate lymphocyte proliferation and IL-2 and IFN-gamma production than EL-4 (P < 0.05); while EL-4/SEA-TM + mB7.1-GPI showed a further increased ability than EL-4/mB7.1-GPI and EL-4/SEA-TM in stimulating lymphocyte proliferation and cytokine production in vitro (P < 0.05). Volume of tumor was smaller and survival time of mice was longer in EL-4/mB7.1-GPI vaccine group, EL-4/SEA-TM vaccine group and EL-4/SEA-TM + mB7.1-GPI vaccine group, comparing with PBS group and EL-4 cell vaccine group (P < 0.05). Tumor volume was much smaller and survival time of mice was much longer in EL-4/mB7.1-GPI + mB7.1-GPI vaccine group, comparing with EL-4/SEA-TM vaccine group and EL-4/mB7.1-GPI vaccine group (P < 0.05). Lymphocytes derived from the mice treated with EL-4/SEA-TM + mB7.1-GPI showed much higher NK activity and CTL activity than those derived from EL-4/mB7.1-GPI vaccine group and EL-4/SEA-TM vaccine group (P < 0.05), meanwhile the NK activity and CTL activity of EL-4/mB7.1-GPI vaccine group and EL-4/SEA-TM vaccine group was higher than EL-4 vaccine group (P < 0.05). CONCLUSION: mB7.1-GPI or/and SEA-TM fusion protein was stably anchored onto the surface of EL-4 tumor cells. The tumor cell vaccines prepared from these cells exhibited antitumor effect. The mB7.1-GPI and SEA-TM dual-anchored tumor cell vaccine had much stronger antitumor effect than the single-anchored tumor cell vaccine.