Curcumin Administered in Combination with Glu-GNPs Induces Radiosensitivity in Transplanted Tumor MDA-MB-231-luc Cells in Nude Mice

Curcumin is a type of plant polyphenol extracted from Curcuma longa L. rhizome, which demonstrates antitumor activity in breast cancer cells in vitro. To investigate the combined effect and possible mechanism of curcumin and glucose-gold nanoparticles (Glu-GNPs), the radiosensitivity of breast carcinoma xenografts was assessed in nude mice. MDA-MB-231 cells labeled with firefly luciferase were inoculated into the mammary fatty pads of nude mice to establish a transplantation tumor model of human breast cancer. The tumor-bearing mice were treated with different drugs (curcumin, Glu-GNPs, and cisplatin) for 3 weeks prior to radiotherapy. The body weights and tumor volumes of the mice were measured in regular intervals. Tumor bioluminescence intensity was determined in real-time using an in vivo bioluminescence imaging system to monitor tumor growth. Transplanted tumor tissue samples were taken for hematoxylin and eosin (HE) staining, and the expression of VEGF, HSP90, HIF-1α, and MMP9 was evaluated via reverse transcription-quantitative PCR or immunohistochemistry. The results revealed that the breast tumor-bearing nude mouse model was successfully established, as evidenced by a stable expression of luciferase. Curcumin inhibited the growth of tumors without causing significant weight loss in mice. Furthermore, additive inhibition was demonstrated when curcumin was administered in combination with Glu-GNPs and irradiation. Tumor bioluminescence intensity was decreased in the model group following curcumin, Glu-GNPs, and irradiation treatment. HE staining demonstrated that transplanted tumors were malignant, with necrotic tissue exhibited centrally. It was concluded that curcumin administered in combination with Glu-GNPs and X-ray irradiation could reduce the protein expression of VEGF, HSP90, HIF-1α, and MMP9 in tumor tissue when compared with the model group. Curcumin and Glu-GNPs administered with X-ray irradiation significantly inhibited tumor growth and induced radiosensitivity, which may be associated with the inhibition of angiogenesis in tumor tissue.

Vitamin K2 Inhibits Hepatocellular Carcinoma Cell Proliferation by Binding to 17β-Hydroxysteroid Dehydrogenase 4

Our previous studies have proved that 17β-hydroxysteroid dehydrogenase 4 (HSD17B4) is a novel proliferation-promoting protein. The overexpression of HSD17B4 promotes hepatocellular carcinoma (HCC) cell proliferation. Vitamin K2 (VK2), a fat-soluble vitamin, has the function of promoting coagulation and can inhibit the progression of liver cancer. A previous study demonstrated that VK2 could bind to HSD17B4 in HepG2 cells. However, the mechanism of VK2 in inhibiting HCC cell proliferation is not clear. In this study, we investigate whether VK2 can inhibit the proliferation of HCC cell induced by HSD17B4 and the possible mechanism. We detected the effect of VK2 on HSD17B4-induced HCC cell proliferation, and the activation of STAT3, AKT, and MEK/ERK signaling pathways. We measured the effect of HSD17B4 on the growth of transplanted tumor and the inhibitory effect of VK2. Our results indicated that VK2 directly binds to HSD17B4, but does not affect the expression of HSD17B4, to inhibit the proliferation of HCC cells by inhibiting the activation of Akt and MEK/ERK signaling pathways, leading to decreased STAT3 activation. VK2 also inhibited the growth of HSD17B4-induced transplanted tumors. These findings provide a theoretical and experimental basis for possible future prevention and treatment of HCC using VK2.

The Molecular Mechanism of ESZM Extract on Treatment ER(-) Breast Cancer in Vivo: Application of “Combat Poison with Poison” Theory

Background: Extract of Euphorbia fischeriana S. and Ziziphus jujuba M. (ESZM) is a formula preparation, was composed of Euphorbia fischeriana S. (E. fischeriana) and Ziziphus jujuba M. (Z. jujuba). The aim of the current research was to examine for the first time the pharmacological effects and the underlying molecule mechanism of ESZM extract on the growth inhibition and apoptosis in human ER(-) or ER(+) breast cancer in vivo, and enrich and innovate the theory of traditional Chinese medicine of combating poison with poison. Methods: Growth inhibition, cell cycle arrest and apoptosis in tumor tissues were determined by tumor inhibition rates, cycle analysis kit, Annexin V-FITC/PI staining, Hoechst 33342/PI staining, TUNEL test and TEM observation. Related genes and proteins expression levels were identified by qRT-PCR assay, Western Blotting method, immunohistochemistry and immunofluorescence means. Drug toxicity was analysised by serum biochemistry detection and H&E staining.Results: We found that ESZM extract can inhibite growth, block cell cycle at G2/M phase and induce apoptosis of subcutaneously transplanted tumor, especially ER(-) breast cancer transplanted tumor. Furthermore, we demonstrated that the polyjuice can down-regulate or up-regulate the expression of Bcl-2 family and PI3k/Akt pathway -related signaling molecules proteins and genes in vivo. ESZM extract or E. fischeriana extract increased the levels of ALT, AST, Cr and BUN, and increased liver and kidney toxicity in tumor-bearing mice by serum biochemistry detection and H&E staining.Conclusion: The pharmacological detection suggested that ESZM extract had significant anti-breast cancer effect, especially ER(-) breast cancer xenograft, and this process may be implement through the mitochondrion dependent pathway and the PI3k/Akt signaling pathway. Drug toxicity detection proved that ESZM extract because of compatibility with Z. jujuba was lower toxicity than E. fischeriana extract, was no significant difference in hepatorenal toxicity between ER(-) or ER(+) tumor-bearing mice.

Experimental Research on the Inhibitory Effect of IFN-Lambda 3 Combined with Sorafenib on the Growth of Liver Cancer Transplanted into Nude Mice

Objective: To investigate the effect of sorafenib combined with interferon-lambda 3 on the growth of liver cancer transplanted into nude mice. Methods: Female nude mice of 4-5 weeks of age that passed quarantine were selected and fed for 1-2 weeks before experimental operation. The cell suspension of human hepatoma cell line SMMC-7721 was inoculated into the right cervical axillary fossa with a syringe. The tumor-bearing mice were randomly divided into a control group and an experimental group. The control group received normal saline whereas the experimental group was further divided into three other groups: IFN-lambda 3 treatment group, sorafenib treatment group, and IFN-lambda 3 combined with sorafenib treatment group. The situation of nude mice was analyzed. At the end of the experiment, the volume of allogeneic transplanted tumor was measured, and the morphology of tumor cells, the expression of proliferating protein Ki-67, as well as the number of apoptotic cells were observed by hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and TUNEL staining. Results: The tumor cell volume of the IFN-lambda 3 treatment group, sorafenib treatment group, and IFN-lambda 3 combined with sorafenib treatment group decreased, which was statistically significant compared with the control group (p < 0.05). The increment rate of proliferating protein Ki-67 in the transplanted tumor tissue of the three drug groups was significantly lower than that of the control group (p < 0.05). IFN-lambda 3 combined with sorafenib had the greatest effect on the expression level of Ki-67 protein. Compared with the control group, the expression rate was significantly lower (p < 0.05). In terms of cell apoptosis, IFN-lambda 3 and/or sorafenib, as well as the combination of the two, showed statistically significant differences compared with the control group (p < 0.05). The rate of cell apoptosis was the highest in the IFN-lambda 3 combined with sorafenib group. Conclusion: IFN-lambda 3 combined with sorafenib can inhibit the growth and proliferation of human hepatocellular carcinoma cells in nude mice and promote the apoptosis of hepatocellular carcinoma cells, which proves that IFN-lambda 3 combined with sorafenib can treat hepatocellular carcinoma in vivo.

Study on Molecular Mechanism of Benzo (ɑ) pyrene on CMA by HSP90ɑ and HIF-1ɑ

OBJECTIVE: The effects of benzo (α) pyrene (BaP) on molecular chaperone autophagy (CMA) through heat shock protein 90 (HSP90) and hypoxia inducible factor-1 (HIF-1) were studied by RNA interference and subcutaneous tumor formation technique in nude mice. METHODS: 40 nude mice inoculated with the shHSP90ɑ A549 cell line under the armpits of the forelimbs were divided into 4 groups, 1.80 mg/kg/d BaP-corn oil solution was intragastrically administered for 60 days (except the Control group), and the growth of nude mice and transplanted tumors was recorded curve. The size and morphological changes of tumors were observed by small animal imaging technique and HE staining method. qPCR, Western blot and Immunohistochemistry were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. A549 cells were treated with 0.1μmol/L, 1μmol/L and 10μmol/L BaP for 24h, EPO, HIF-1ɑ concentration and HIF-1ɑ protein expression were detected by Elisa and Western blot; A549 cells were treated with 10μmol/L BaP and added HIF-1ɑ inhibitor 24h, qPCR, Western blot and Immunofluorescence method were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. RESULTS: BaP can reduce silence HSP90ɑ the weight of nude mice and transplanted tumors (P<0.01); BaP can reduce silence HSP90ɑ the expression of HSP90ɑ, HSC70, Lamp-2A mRNA and protein in transplanted tumor tissues (P<0.05); BaP has no significant difference in the organization structure of silence HSP90ɑ and non-silence HSP90ɑ; BaP can reduce the total number of bioluminescence photons of silence HSP90ɑ transplanted tumors (P<0.01). 10μmol/L BaP can increase the concentration of EPO and HIF-1ɑ and the expression of HIF-1ɑ protein in A549 cells (P<0.05); and after adding HIF-1ɑ inhibitors treat A549 cells, HSP90ɑ, HSC70, Lamp-2A mRNA and protein expression were decreased (P<0.05), and the fluorescence intensity of HSP90ɑ was decreased. CONCLUSIONS: BaP can promote the growth of transplanted tumor in nude mice, while the growth of transplanted tumor in nude mice is inhibited shHSP90ɑ. BaP promotes the occurrence of CMA by promoting the expression of HSP90ɑ and HIF-1ɑ, which are important regulatory genes for BaP to activate CMA.

Analysis of Molecular Mechanism of YiqiChutan Formula Regulating DLL4-Notch Signaling to Inhibit Angiogenesis in Lung Cancer

In order to explore the specific mechanism of YiqiChutan formula (YQCTF) in inhibiting the angiogenesis of lung cancer and its relationship with delta-like ligand 4- (DLL4-) Notch signaling, 30 healthy BALB/c-nu/nu rats were selected and divided into three groups: A549 group (implanted with lung adenocarcinoma cell line A549), NCI-H460 group (implanted with human lung large-cell carcinoma cell line NCI-H460), and NCI-H446 group (implanted with human lung small cell carcinoma cell line NCI-H446) for constructing lung cancer transplanted tumor models. After modeling, the group treated with normal saline was taken as control group, 200 mg/kg of YQCTF was adopted for intervention, and the tumor volume and growth inhibition rate were compared with the vascular targeted inhibitor Sorafenib. HE staining, CD31 fluorescent antibody staining, and microelectron microscopy were adopted to observe the neovascular endothelial cells of the transplanted tumor. The expression of VEGF, HIF-1α, DLL4, and Notch-1 in the transplanted tumors in each group was detected by Western blot and RT-PCR at the protein level or mRNA level. Compared with the control group, the YQCTF-treated group had obvious inhibitory effect on lung cancer transplanted tumor and lung cancer angiogenesis. In the YQCTF-treated group, the density of angiogenesis decreased significantly and the vascular lumen structure also decreased, and the expression levels of VEGF, HIF-1α, DLL4, and Notch-1 in the YQCTF-treated group were all lower than those in the control group. YQCTF could inhibit the growth of lung cancer transplanted tumor through antiangiogenesis, and it could also reduce the amount of angiogenesis in lung cancer transplanted tumor. In addition, the generation of lumen structure was also hindered, which was realized through the VEGF signaling pathway and DLL4-Notch signaling pathway.

Experimental model and approaches to investigation of the acquired resistance to tumor transplantation in mice

Introduction. An acquired resistance to experimental tumors was detected in animals that recovered from a primary transplanted tumor due to treatment or spontaneously, and demonstrated intolerance to a renewal tumor inoculation. This phenomenon is much less frequently observed, although it is of great scientific interest and medical significance. Here, we have addressed the expression of the resistance phenomenon in a model tumor in mice – Nemeth–Kelner lymphoma (NK/Ly). The aim of our study was to elaborate a reproducible method for induction of resistance to transplantation of lymphoma NK/Ly in mice and to investigate the mechanisms of its development. Methods and Results. Three schemes for induction of resistance were tested. The first one included treatment of tumor-bearing mice with vinblastine and, thereafter, reconvalescent animals were checked for the development of resistance expressed as a complete suppression of tumor growth after re-inoculation of tumor cells. Mice were inoculated intraperitoneally with NK/Ly ascitic cells and then subjected to 2–4 intraperitoneal injections of vinblastine at a dose of 1mg/g of body weight. The recovered mice were re-inoculated with tumor cells and the absence of tumor growth was considered as resistance development. The disadvantage of this approach is that less than 5% of mice achieve a long lasting recovery due to the treatment. The second scheme included the immunization of mice with intraperitoneal injection of the minimal number of viable tumor cells that do not cause tumor growth, but initiate the immune response. However, this approach was not effective, since there was no reliable number of cells correspon­ding to these demands. The minimal number of 15×103 injected cells per mouse caused a retarded but still progressive tumor growth. In the third scheme, the immunization of mice was conducted by the intraperitoneal injections of NK/Ly cells permeabilized with saponin. It should be noted that treatment with saponin leads to cell death with a minimal damage to cell morphology. The scheme of immunization with permeabilized NK/Ly cells appeared to be simple and effective. It provided a reproducible resistance to transplanted tumor and might be used as a model in studies of the mechanisms of this phenomenon. Cytological investigation of tumor and immunocompetent cells in ascites of control and of tumor-resistant mice was conducted. As revealed, the number of lymphocytes in ascites of tumor-resistant mice was about 4 times higher than such amount in the control (non-resistant) mice. A destruction of tumor cells by the adherent mono-nuclears was observed. Conclusions. The method of induction of resistance to transplantation of experimental tumor NK/Ly by immunization of mice with tumor cells permeabilized with saponin is described. The intraperitoneal inoculation of tumor cells to the tumor-resistant mice caused the marked increase of the mononuclear leukocytes population in the peritoneal fluid, which showed a harmful effect upon tumor cells. Thus, the induction of resistance to transplantation of NK/Ly lymphoma in mice might be provided mainly via the mechanisms of cell immunity, in particular, by the appearance of cytotoxic lymphocytes specific to distinct tumor cells.

Solanine Inhibits Immune Escape Mediated by Hepatoma Treg Cells via the TGFβ/Smad Signaling Pathway

Objective. To observe the inhibitory effect of solanine on regulatory T cells (Treg) in transplanted hepatoma mice and to study the mechanism of solanine inhibiting tumor growth. Methods. The levels of Treg cells and IL-2, IL-10, and TGFβ in the blood of patients with liver cancer were detected by flow cytometry and ELISA, respectively. A mouse hepatocellular carcinoma (HCC) graft model was established and randomly divided into four groups: control group, solanine group, TGFβ inhibitor group (SB-431542), and solanine +TGFβ inhibitor combined group. Tumor volume of each group was recorded, tumor inhibition rate was calculated, and tumor metastasis was counted. The proportion of CD4+CD25+Foxp3+ Treg in transplanted tumor tissues was detected by flow cytometry. The expression levels of Foxp3 and TGFβ in transplanted tumor tissues were detected by quantitative fluorescence PCR. Results. Compared with healthy people, Treg cells and IL-2, IL-10, and TGFβ contents in peripheral blood of liver cancer patients were increased. The results of the transplanted tumor model in mice showed that the tumor volume of the transplanted mice in the solanine group and the TGFβ inhibitor mice was reduced compared with the control group. The combined group had the smallest tumor volume. The proportion of CD4+CD25+Foxp3+ Treg in the transplanted tumor tissues of mice in the solanine treatment group was significantly lower than that in the control group. The expressions of Foxp3 and TGFβ in the transplanted tumor tissues of mice in the solanine group were significantly lower than those in the control group. Conclusion. Solanine may enhance the antitumor immune response by downregulating the proportion of CD4+CD25+ Treg and the expression of Foxp3 and TGFβ in tumor tissues.

Double-Targeted Knockdown of miR-21 and CXCR4 Inhibits Malignant Glioma Progression by Suppression of the PI3K/AKT and Raf/MEK/ERK Pathways

Currently, miR-21 and CXCR4 are being extensively investigated as two key regulators in glioma malignancy. In this study, we investigated the combined effects of these two factors on glioma progression. Herein, the expression of miR-21 and CXCR4 was increased in tumor tissues and cell lines. Inhibition of miR-21, CXCR4, and miR-21 and CXCR4 together all reduced the migration, invasiveness, proliferation, and enhanced apoptosis in glioma cells, as well as reduced tumor volume and mass in xenograft model. The inhibition effect was strongest in double-targeted knockdown of miR-21 and CXCR4 group, whose downstream pathways involved in AKT axis and ERK axis activation. In conclusion, our findings reported that double-targeted knockdown of miR-21 and CXCR4 could more effectively inhibit the proliferation, migration, invasion, and growth of transplanted tumor and promote cell apoptosis, which were involved in the PI3K/AKT and Raf/MEK/ERK signaling pathways.