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  • br RNA was extracted from whole cell

    2020-08-28


    RNA was extracted from whole cell lysates and reverse transcribed to cDNA using a reverse-transcriptional kit (TaKaRa). RT-PCR was performed in a triplicate with the SYBR Prime Script RT-PCR kit (TaKaRa) on the Step One Plus Real-Time PCR system (Applied Biosystems). The sequences are described in the Supporting Information for Materials and Methods.
    2.11. Western blot analysis
    Lysates were prepared from tumor tissues and Erlotinib and then cen-trifuged at 14,000 rpm for 15 min. Protein concentration in each cell lysate was determined using BCA assay kit, and all samples were elec-trophoresed through 10% SDS-PAGE gel and transferred to PVDF membranes. The blots were probed with appropriate primary anti-bodies. After incubation with secondary antibodies, membranes were washed and stained with ECL according to the manufacturer’s protocol.
    2.12. Statistical analysis
    Data are expressed as mean ± SEM. Each value is the mean of at least three separate experiments in each group. The statistical sig-nificance between groups was determined by the Student’s t-test or one-way ANOVA using the GraphPad Prism 6.0 software (GraphPad Software Inc., USA). P < 0.05 was considered as the statistically sig-nificant difference.
    3. Results
    3.1. Blocking the sympathetic signaling reduces the release of catecholamines and delays tumor growth
    To investigate the interplay between adrenergic nerves and cancer growth, BALB/c nu/nu mice were injected with human non-small cell lung cancer cells HCC827 in the right upper flanks or small cell lung cancer cells H446 in the right hind limbs. These mice of each cancer model were randomly divided into two groups: the control group in-jected with PBS, and Erlotinib the experimental group chemically sympathecto-mized with 6OHDA to suppress secretion of catecholaminergic neuro-transmitter in vivo. In both the H446 and HCC827 xenograft models, tumor development was delayed significantly, with mice in the 6OHDA group having smaller tumor sizes and lower tumor weights (Fig. 1A). On the 24th day, the tumor weights of control groups were 4.61 and 2.30-fold in H446 mice and in HCC827 mice, respectively, greater than those in the 6OHDA groups. To confirm the connection between tumor growth and the level of catecholamines, we assessed the concentration of EPI and NE in removed lungs, spleens, and tumors by ELISA. The results showed significant reductions in levels of catecholamines in 6OHDA group mice compared with controls (Fig. 1B). Consistent with the secretion characteristics of catecholamines, we found that the in-hibited extent of transmitters varied with tissues. Although the down-trend of EPI and NE were in synchrony, the release of EPI was pre-dominantly suppressed in spleens and lungs, while NE release was largely inhibited in tumor tissues. Especially, the relative NE con-centrations of 6OHDA groups were 16.0% and 31.3% in H446 mice and
    Y. Xia, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
    Fig. 1. Decreasing catecholamine levels delays lung cancer growth. (A) Subcutaneous inoculation with H446 cells (upper, left) and HCC827 cells (upper, right) was established in mice and observed using in vivo imaging. The tumor volume was measured for 2 weeks consecutively after injecting H446 xenograft (lower, left) or HCC827 xenograft (lower, right) mice with 6OHDA; n = 5 or 6. Representative pictures of xenograft models are shown. (B) Tumor tissues, spleens, and lungs were removed from mice to detect the concentrations of EPI and NE. n = 3. *p < 0.05. **p < 0.01. ***p < 0.001. Abbreviations: N.S.: non-statistical significance.
    in HCC827 mice, respectively, lower than those in control groups. However, similar levels of EPI were observed in H446 (P = 0.9738) or HCC827 (P = 0.2905) tumor tissues. EPI comes from the adrenal gland predominately, and may reach the tumor through blood circulation. In general, the only catecholamine released into the tumor micro-environment directly by SNS nerves would be NE. That may be the reason that why the decline of NE in tumor tissues is even more 
    pronounced than EPI after 6OHDA treatment. There were also some interesting differences between H446 and HCC827 models for the former achieved a greater inhibition of tumor growth with a lower NE concentrations in tumors, spleens and lungs. These results indicate that peripheral adrenergic nerves might positively play a role in regulating tumor progress, by releasing catecholaminergic neurotransmitters, through which the effect mediated by NE might be dominant in tumors.
    Y. Xia, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
    Fig. 2. Reduction of catecholamine levels affects macrophage polarization. (A) Spleens (upper) and tumor tissues (lower) were removed to detect the activation of macrophages using flow cytometry after 6OHDA injection. n = 3. (B) Typical cytokines secreted by activated macrophages were tested in tumors. *p < 0.05. **p < 0.01. Abbreviations: N.S.: non-statistical significance.