Effects of Chlorella vulgaris on tumor growth in mammary tumor-bearing Balb/c mice: discussing association of an immune-suppressed protumor microenvironment with serum IFNγ and IgG decrease and spleen IgG potentiation.

PURPOSE: Chlorella vulgaris (CV) has exhibited immune-enhancing and protective activities against cancer and infections. However, there is an increasing concern about the use of Chlorella species in human, regarding its various molecules with antigenic features found in infectious microorganisms. Our goal was to investigate the impact of higher concentrations of CV on tumor growth in spontaneous mouse mammary tumor (SMMT) models. METHODS: Balb/c mice were daily given CV powder at doses of 0, 200, or 300 mg/kg for 42 days (CONTROL, CV200, and CV300 groups, respectively; n = 6/group). On day 14, the SMMT was inoculated. Tumor volume (TV) and body weight (BW) were monitored on 5-day intervals following tumor challenge. On day 43, blood, spleen, lungs, and tumor tissues were collected. Histopathological examinations on lungs and tumor tissues were performed following hematoxylin-eosin staining. Intratumor expression of 27 genes was assessed by real-time PCR. Total IgG, IFNγ, and IL-4 levels in serum and spleen culture supernatant were measured by ELISA. RESULTS: The TV/BW index showed significant increase in the CV200 group compared to the CONTROL (p = 0.047). The CV200 tumors exhibited more malignant phenotype, higher angiogenesis rate, and lower peritumoral neutrophil and macrophage-to-lymphocyte infiltration ratio compared to the CONTROL. Serum concentrations of IFNγ, IL-4, and IgG were declined, and the spleen IFNγ and IgG production was higher in the CV200 compared to the CONTROL. The IL-1ß, IL-10, TGFß1, FOXP3, HO-1, Gr1, CD11b, PCNA, LCN2, iNOS2, VEGFR2, CD31, and CD105L expressions were markedly increased in the CV200 tumors compared to the CONTROL (p = 0.001, 0.002, 0.006, 0.021, 0.004, 0.030, 0.016, 0.031, 0.025, 0.008, 0.014, 0.022, and 0.037, respectively). The changes in cytokine, IgG and gene expression values considerably correlated with tumor size, as well as with each other. CONCLUSIONS: Our data provided evidence that C. vulgaris at a specific dose (200 mg/kg) promoted tumor growth in a mammary tumor model. This consequence might reflect an immune derangement in favor of developing a protumor microenvironment. However, this hypothesis needs to be further investigated in future.

Spontaneous mouse mammary tumor cell lysates induce IgG production in spleen mononuclear cells of healthy and tumor-bearing mice.

BACKGROUND: We hypothesized that Tumor cell lysate (TCL) prepared from spontaneous mouse mammary tumor (SMMT) may elicit IgG production by spleen mononuclear cells (SMCs) in ex vivo. METHODS: The SMCs from healthy mice (HM, n = 6) and four-week SMMT-bearing mice (TBM, n = 6) was cultured in presence of TCL and mitogen for 42 hr at 37°C, separately. Serum and SMCs culture supernatant levels of IFNγ, IL-4, and total IgG were measured using special ELISA kits. RESULTS: Serum IgG level of TBM was significantly higher than that of HM group (P = 0.019), while serum IFNγ and IL-4 did not differ between two groups (P > 0.05). Mitogen significantly induced ex vivo production of both IFNγ (P = 0.013) and IL-4 (P = 0.015) by SMCs from HM group, and only IL-4 (P = 0.049) by SMCs from TBM group. In contrast, TCL increased ex vivo production of IgG by SMCs from both HM (P = 0.034) and TBM (P = 0.016) groups. The ex vivo IgG revealed a moderate positive correlation with tumor size (r = 0.578, P = 0.422). CONCLUSION: It seems that TCL prepared from SMMT are potent inducers of IgG production. This may propose TCL as a potential tool for monitoring of humoral immunity in animal models.

Harmaline downregulates angiogenesis markers and suppresses the growth of 4T1 breast cancer cells in vivo and in vitro.

The anti-angiogenic effects of harmaline, an alkaloid with emerging anti-tumor properties, are under investigation. In the present study, the effects of different doses of harmaline, either alone or in combination with doxorubicin (DOX), were assessed in mice models of breast tumor. Breast tumors were created by the subcutaneous injection of 4T1 cells into Balb/c mice. The mice received either normal saline, harmaline alone (10, 20, or 30 mg/kg), or harmaline (20 mg/kg) + DOX (10 mg/kg). Immunohistochemistry, ELISA, and real-time PCR were conducted to measure target parameters. Harmaline significantly increased tumor cells' sensitivity to DOX as confirmed by a significantly reduced tumor volume in the harmaline + DOX group after 24 days (P < 0.05). Also, the levels of Ki-67 (P < 0.001), MMP-2 (P < 0.001), and VEGF (P < 0.001) significantly decreased while the level of E-cadherin increased (P < 0.001) in the tumor tissues of the mice treated with 20 or 30 mg/kg harmaline or harmaline (20 mg/kg) + DOX (10 mg/kg) compared to the control group. There was a significant reduction in the serum level of IL-4 in tumor-bearing mice treated with harmaline (P < 0.05), and IFN-γ serum level was significantly augmented in all experimental groups compared to the control group (P < 0.05). The genes encoding VEGF, VEGF receptor 2, CD105, and COX2 were significantly down-regulated (P < 0.05 for all) in harmaline-treated (either alone or in combination with DOX) mice. In conclusion, harmaline seems to have the potential to be used as an anticancer agent for treating breast cancer.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer.

OBJECTIVES: Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. METHODS: In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. RESULTS: Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 µM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNÉ£). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. CONCLUSIONS: Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

Thioredoxin-1, redox factor-1 and thioredoxin-interacting protein, mRNAs are differentially expressed in Multiple Sclerosis patients exposed and non-exposed to interferon and immunosuppressive treatments.

BACKGROUND: Oxidative stress is closely linked to inflammation in neurodegenerative diseases. We aimed to investigate the expression of redox system genes in Multiple Sclerosis (MS) patients either exposed or not exposed to conventional treatments. METHODS: Forty-four MS patients were divided into three groups: newly diagnosed (Group 1), receiving interferon (Group 2) and receiving immunosuppressive drugs (Group 3). Also, 15 healthy controls were enrolled. The mRNA expression of TRX1, TXNRD1, TRX2, TXNRD2, TXNIP, and APEX1 genes in peripheral blood mononuclear cells (PBMCs) was assessed by relative quantitative real-time PCR. Also, serum level of Trx1 was measured by ELISA. RESULTS: Serum level of Trx1 in the newly diagnosed MS patients was significantly higher compared to the healthy controls (P=0.013). Likewise, TRX1 and APEX1 expressions were significantly higher in the newly diagnosed patients compared to controls (P=0.003 and P=0.042), patients under interferon treatment (P=0.003 and P=0.013), and patients received immunosuppressants (P=0.001 and P=0.025). Furthermore, TXNIP expression in MS patients (either group 1, group 2, or group 3) was significantly lower than that in the control group (P=0.017, P=0.002, and P=0.022 respectively). The expression of TXNRD1, TRX2, and TXNRD2 did not show any significant difference between the control and the MS patient (P>0.05). CONCLUSIONS: Our data showed that redox system elements are differentially expressed in newly diagnosed MS patients, or patients receiving either interferon or immunosuppressive treatments. However, much more studies are required to confirm our findings and clarify the underlying mechanisms.