Beclin1 inhibition enhances paclitaxel­mediated cytotoxicity in breast cancer in vitro and in vivo.

Beclin1, a key regulator of autophagy, has been demonstrated to be associated with cancer cell resistance to chemotherapy. Paclitaxel is a conventional chemotherapeutic drug used in the clinical treatment of breast cancer. However, the function and mechanism of Beclin1 in paclitaxel­mediated cytotoxicity in breast cancer are not well defined. The present study demonstrated that paclitaxel suppressed cell viability and Beclin1 expression levels in BT­474 breast cancer cells in a dose­ and time­dependent fashion. Compared with the control, the knockdown of Beclin1 significantly enhanced breast cancer cell death via the induction of caspase­dependent apoptosis following paclitaxel treatment in vitro (P<0.05). In a BT­474 xenograft model, paclitaxel achieved substantial inhibition of tumor growth in the Beclin1 knockdown group compared with the control group. Furthermore, analysis of the publicly available Gene Expression Omnibus datasets revealed a clinical correlation between Beclin1 levels and the response to paclitaxel therapy in patients with breast cancer. Collectively, the present results suggest that Beclin1 protects breast cancer cells from apoptotic death. Thus, the inhibition of Beclin1 may be a novel way to improve the effect of paclitaxel. Additionally, Beclin1 may function as a favorable prognostic biomarker for paclitaxel treatment in patients with breast cancer.

BECN1-knockout impairs tumor growth, migration and invasion by suppressing the cell cycle and partially suppressing the epithelial-mesenchymal transition of human triple-negative breast cancer cells.

Beclin1 (BECN1), which directly interacts with B­cell lymphoma 2, serves an important role in autophagy and is involved in the tumorigenesis of various types of cancer. However, the definite role of BECN1 in breast cancer remains controversial. Bi-allelic knockout of Becn1 in a mouse model leads to an embryonic lethal phenotype, which hampers further investigation. To generate cell lines with knockout of BECN1, the CRISPR/Cas9 technique was used to disrupt BECN1 in human triple-negative breast cancer (TNBC) MDA­MB­231 cells. To the best of our knowledge, the present study was the first to successfully disrupt BECN1 in MDA­MB­231 cells and to screen three stable monoclonal BECN1­knockout cell lines, suggesting that BECN1­knockout is not lethal in TNBC cells. Functional analysis revealed that complete loss of BECN1 suppressed MDA­MB­231 proliferation and colony formation via inducing G0/G1 cell cycle arrest, not apoptosis, in vitro. On the other hand, BECN1­knockout inhibited the migratory and invasive ability of MDA­MB­231 cells by partially reversing signals of epithelial-mesenchymal transition. Finally, analysis of publicly available gene expression datasets revealed increased expression of BECN1 in TNBC samples. Taken together, the results of the present study identified BECN1 as an oncogene, providing a novel potential target for the treatment of TNBC.

[Preparation and characterization of monoclonal antibody against Vibrio vulnificus].

AIM: To prepare high-performance and specific monoclonal antibody (mAb) against Vibrio vulnificus and carry out characterization. METHODS: BALB/c mice were immunized with Vibrio vulnificus protein, and hybridoma cells against Vibrio vulnificus were produced by cellsion technique. The titers of mAbs against vvhA and cross-reaction between the anti-vvhA mAb and other other important marine bacteria were screened by ELISA and Western blot. RESULTS: Five strains of hybridoma were obtained. Identification result indicated that 5 mAbs have favourable specifity and immunoreactivity. CONCLUSION: Specific mAbs against Vibrio vulnificus were produced which provides an important preparation for establishing rapid-detection kit of detecting Vibrio vulnificus.

[Jak/STAT signaling pathway of IL-11 in the protection of intestinal epithelial cells from neutron radiation].

AIM: To explore the effect of IL-11 on the activation of Jak/STAT pathway and the expressions of Bax and Bcl-2 in the intestinal epithelial cells exposed to neutron radiation. METHODS: The BALB/c mice and IEC-6, irradiated by 4 Gy neutron with or without IL-11 treatment, served as in vivo and in vitro model seperately. The changes of the intestines, activity of Jak1 and STAT3 and expressions of Bax and Bcl-2 were observed by HE staining, Western blot, EMSA, immunohistochemistry and image analysis. RESULTS: (1)Mice exposed to neutron radiation showed severe intestinal damages and no obvious regeneration was seen. IL-11-treated mice had a larger number of cryptal epithelial cells and crypts. (2)Neutron radiation decreased the activities of Jak1 and STAT3, while IL-11 increased their activities. (3) Neutron radiation decreased the expression of Bax and didn't change the level of Bcl-2 in the murine intestine. IL-11 administration decreased the expression of Bax and increased that of Bcl-2. CONCLUSION: The mechanism of the intestinal protection of IL-11 in neutron irradiation might be that IL-11 stimulation triggered activation of Jak/STAT pathway, downregulated the expression of Bax and upregulated the expression of Bcl-2.

[Effect of IL-2 on the growth and apoptosis of intestinal epithelial cells radiated by neutron and mechanisms of IL-2 on the injured IEC-6].

AIM: To observe the effect of neutron radiation on intestinal epithelial cells 6 (IEC-6), to study the effect of IL-2 on the proliferation and recovery of neutron-injured IEC-6, and to investigate the regulatory mechanisms of IL-2 on the injured IEC-6. METHODS: 4Gy-neutron-injured IEC-6 were treated by IL-2, with or without the blocking agent JAK(1) (A77-1726). The change of proliferative activity and death manner of the treated IEC-6 were detected by MTT colorimetry and flow cytometry at 10, 15, 30 minutes and 1, 3, 6, 12, 24, 48, 72 hours respectively. The expression of IL-2Rbeta and the activation of JAK(1) of neutron-injured IEC-6 treated by IL-2 were detected by immunocytochemical stainning and Western blot. RESULTS: After IEC-6 were radiated by 4 Gy neutron for 24 hours, the proliferative activity of IEC-6 decreased markedly but increased strikingly after IL-2 treatment (P<0.05). The apoptosis of IEC-6 in IL-2-treated group decreased (P<0.05), but there was no obvious difference in necrotic cell number. After neutron-injured IEC-6 were treated by IL-2, JAK(1) was activated at 10 and 15 minutes, and the expression of IL-2Rbeta increased apparently at 24 hours. When treated by JAK(1) and IL-2, the proliferative activity of neutron-injured IEC-6 was much lower than that in IL-2-treated group. CONCLUSION: IL-2 can accelerate the proliferation of neutron-radiated IEC-6 and protect them from neutron injury. IL-2Rbeta and JAK(1) participate in the regulation of neutron-injured IEC-6 by IL-2.

Effect of recombinant human interleukin-11 on expressions of interleukin-11 receptor alpha-chain and glycoprotein 130 in intestinal epithelium cell line-6 after neutron irradiation.

AIM: To explore the effect of recombinant human interleukin-11 (rhIL-11) on the expressions of interleukin-11 receptor alpha-chain (IL-11Ralpha) and an additional signal transducer glycoprotein 130 (gp130) in intestinal epithelium cell line-6 (IEC-6) after neutron irradiation. METHODS: Cultured IEC-6 cells were exposed to 4.0Gy neutron and treated with 100 ng/mL rhIL-11 12 h prior to or immediately after irradiation. The apoptosis and necrosis rates and expressions of IL-11Ralpha and gp130 were observed by flow cytometry, immunohistochemistry, Western blot and image analysis. RESULTS: The apoptosis rate of IEC-6 cells was increased by irradiation at 6 h (P < 0.01), IL-11 stimulation resulted in a decreased apoptosis rate in irradiated IEC-6 cells (P < 0.05). In normal control IEC-6 cells, intense immunoreactivity of IL-11Ralpha was located within the cell membrane and cytoplasm. The level of IL-11Ralpha expression significantly decreased at 6 h after irradiation (P < 0.01) and restored at 24 h after irradiation. In IEC-6 cells treated with both radiation and rhIL-11, the level of IL-11Ralpha expression was higher than that of irradiated cells (P < 0.05). When it came to gp130 protein, it was located in the cytoplasm of IEC-6 cells. After irradiation, we found a progressive decrease in the expression of gp130 protein (P < 0.05) in 48 h post-radiation, while in rhIL-11-stimulated cells, it came back to normal level at 24 h after irradiation and decreased at 48 h, but was still higher than that of only irradiated cells (P < 0.05). CONCLUSION: rhIL-11 can protect IEC-6 cells from neutron irradiation. The protective effect of rhIL-11 might be connected with its ability to up-regulate the expressions of specific ligand-binding subunit IL-11Ralpha and signal-transducing subunit gp130.

[The expression of TNF-alpha in the intestine of mice irradiated by neutron and gamma rays].

AIM: To study the expression of tumor necrosis factor alpha (TNF-alpha) in the intestine of mice irradiated by neutron and gamma rays. METHODS: 350 male BALB/c mice were irradiated with neutron and gamma rays of different doses, and sacrificed at 6 and 12 hours, 1, 2, 3, 4, 5, 7, 10, 14, 21 and 28 days after irradiation. The TNF-alpha in the mice intestinal tissue was detected by means of immunohistochemical staining and image analysis. RESULTS: In normal control mice, TNF-alpha was expressed in the cytoplasm of macrophages in intestinal villus interstitium, submucosa and lymph tissue. After 2.5Gy neutron radiation, TNF-alpha was decreased progressively within 2 days, increased obviously in macrophages and crypt cells during the 3rd-7th day, reached the peak at the 5th day and recovered to normal level at the 14th day. TNF-alpha was decreased progressively within 4 days after 4.0 and 5.5Gy neutron and 12Gy gamma ray irradiation. TNF-gamma was increased obviously in 6-12 hours, decreased on the first day, increased at the 2nd-5th day, peaked at the third day and recovered at the 10th day after 5.5Gy gamma ray irradiation. CONCLUSION: Neutron and gamma ray radiation induce different expression profile of endogenous TNF-gamma in small intestine, which may be related with the pathologic courses of irradiation-induced damage and repair of intestine.

[An experimental study of the effect of IL-2 on the growth of irradiated intestinal epithelial cells].

AIM: To observe the effect of ionizing radiation on intestinal epithelial cells 6 (IEC-6) and the effect of IL-2 on the proliferation and recovery of radiation-injured IEC-6 cells. METHODS: IEC-6 cells exposed to 4, 8 and 12 Gy gamma ray were analyzed with MTT colorimetry, light microscopy, electron microscopy, DNA gel electrophoresis and flow cytometry at 3, 6, 9, 12 hours and 1, 2, 3 days after radiation. Proliferation of 8 Gy gamma ray-irradiated IEC-6 cells which was treated with IL-2 was detected by MTT colorimetry at 3, 6, 9, 12 and 24 hours after irradiation. RESULTS: Proliferation of IEC-6 decreased with the increased dose of gamma ray radiation. Apoptotic IEC-6 cells increased apparently at 24 hours after radiation with 8.0 Gy gamma ray, IL-2 could enhance the proliferation of irradiated IEC-6 cells (especially at concentration of 1 x 10(5) U/L) in a dose-dependent manner. CONCLUSION: 4-12 Gy of gamma ray radiation can reduce the proliferation and cause apoptosis of IEC-6 cells. IL-2 can accelerate proliferation of irradiated IEC-6 cells and protect IECs from ionization radiation injury.