Cisplatin (also known as diaminedichloroplatinum, DDP) is currently one of the most effective chemotherapeutics for treatment of ovarian cancer. However, the resistance to DDP is a common obstacle. The mechanisms of DDP resistance are unclear. Copper chaperone for superoxide dismutase1 (CCS) mediates the transfer of Cu2+ to superoxide dismutase 1 (SOD1), thereby maintains cell proliferation and survival. Contrarily, inhibition of CCS can retard tumor cell proliferation. This study aims to demonstrate that AMPKdependent CCS expression is involved in ovarian cancer DDP resistance. Realtime quantitative PCR (RT-qPCR) and Western blotting results showed that the levels of CCS mRNA and protein in DDP-resistant ovarian cancer C13* cells were markedly up-regulated when compared to that of DDP-sensitive OV2008 cells. Silencing CCS by shRNA or inhibiting CCS by the inhibitor DC_AC50 could significantly enhance DDP-inhibited proliferation of C13* cells, indicating that high expression of CCS is associated with DDP resistance, and suppression of CCS can reverse DDP resistance. Similarly, Western blotting results revealed that the levels of CCS in six lung cancer cells including A549 and H1944 were closely correlated to the sensitivities of cancer cells to DDP. Silencing CCS expression markedly increased DDP sensitivity of A549, while overexpression of CCS decreased DDP sensitivity of H1944, which further demonstrated the correlation of CCS with DDP resistance. In addition, blocking the phosphorylation/activation of AMPKα at Thr172 by Compound C, an inhibitor of AMPK, could down-regulate CCS expression in C13* and enhance its sensitivity to DDP. These data suggest that AMPK signal-dependent CCS expression is involved in the mechanism of DDP resistance in cancers, and suppressing CCS reverses DDP resistance. These data also indicate that the CCS is hopeful to be a potential target for overcoming DDP resistance.
The orexin 1 receptor (OX1R) and cholecystokinin 2 receptor (CCK2R), which are closely correlated to cellular proliferation, are highly expressed in colon cancer cells. Its mechanism, however, is unclear. Based on the previous finding that OX1R and CCK1R can form dimer in HT-29 cells, in this study we investigate whether OX1R and CCK2R may heterodimerize to function in living cells by using a variety of resonance energy transfer techniques and coimmunoprecipitation (Co-IP). The bioluminescence resonance energy transfer (BRET) signals (BRET ratios) of OX1R and CCK2R in HEK293T cells were analyzed after the cells were exposed to orexin or gastrin for 5 minutes. Results showed that compared with non-exposed (control) cells, BRET signals were increased and reached a maximum when increased the acceptor (CCK2R-eYFP) expression without changes in donor (OX1R-Rluc) expression. The resonance energy transfer signals between OX1R-eYFP and CCK2R-eCFP over-expressed in HEK293T cells were detected by fluorescence resonance energy transfer (FRET) method. Moreover, receptor photobleaching FRET (apFRET) revealed that the FRET efficiency (FRETe) of the donor protein (CCK2ReCFP) in the OX1R-eYFP and CCK2R-eCFP co-transfected living cells was significantly increased to 3.7 folds of that in transfected control cells (P<0.05) after complete photobleaching which disrupted the receptor protein (OX1R-eYFP) and receptor-donor interaction. In addition, the combination of gene transfection and co-immunoprecipitation (Co-IP) indicated that the HA-OX1R and Myc-CCK2R fusion proteins were detectable only in the immunoprecipitates from HAOX1R and Myc-CCK2R co-transfected cells but not in that from transfected control and/or Myc-CCK2R- transfected cells. These dates suggest that OX1R can interact with CCK2R in living cells, which may provide new clues for further investigating the role of OX1R-CCK1R interaction in colon cancer cell proliferation and the relative signaling pathways.
Progranulin (PGRN) is overexpressed in a variety of tumors. However, little is known presently about the function of PGRN in the progression of melanoma. To fill this gap of knowledge, we established a mouse melanoma B16 cell line named B16-PGRNlow with PGRN expression stably knocked down by the CRISPR-Cas9 gene editing technique. MTS and BrdU assays showed that knock-down of PGRN had little effect on B16 cell proliferation or cell cycle status in vitro. We subsequently established the B16 tumor model by subcutaneously injecting B16-ctrl or B16-PGRNlow cells into wild type (WT) and PGRN knockout (KO) mice, respectively. B16-PGRNlow cells displayed significant growth deficiency in both WT and PGRN KO mice (in WT mice, P<0.05; in KO mice, P<0.01) compared with B16-ctrl cells, whereas little difference was observed in tumor growth of WT and KO mice implanted with B16-ctrl cells or with B16 PGRNlow cells. These results indicated that tumor-originated, not host-derived PGRN, is dominate in promoting B16 tumor growth. Last but not least, flow cytometric analysis revealed increased percentage of CD4+and CD8+ T cells in the spleen and tumor-draining lymph nodes in WT mice carrying B16PGRNlow tumor compared with WT mice bearing B16-ctrl tumor, while little difference was found in KO mice carrying either type of tumors. Our study demonstrates for the first time in a definitive manner in vivo that tumor-, not host-derived PGRN promotes B16 melanoma growth. Furthermore, knockdown of PGRN in the tumor results in increased recruitment of CD4+ T and CD8+ T cells in spleen and tumor draining lymph node suggesting the involvement of enhanced cellular immunity of the host. This original study provides insights into the role of PGRN in tumorigenesis and highlights the potential of tumorderived PGRN as a therapeutic target in melanoma.
The solanidine glycosyltransferase (Sgt) family, comprising Sgt1, Sgt2 and Sgt3, participates in the biosynthesis of glycoalkaloids (GAs) in potatoes. It has been shown that inhibition of any one of the Sgt family members influences GA biosynthesis. However, blocking the expression of a single gene of the family does not effectively eliminate the accumulation of total GAs in the tuber. In this study, we employed a strategy to inhibit the expression of Sgt1, Sgt2 and Sgt3 genes simultaneously by RNA interference (RNAi) to reduce the accumulation of GAs in the potato tuber. First, we constructed the RNAi vector pCEI-PFR, in which the expression of siRNAs targeting Sgt1, Sgt2 and Sgt3 genes were driven by the tuber-specific Patatin promoter, and transformed the vector into potato internodal explants by Agrobacterium-mediated transformation. As a result, we obtained ten transgenic plants that contain the Patatin-RNAi fusion gene. Real-time Quantitative PCR (RT-qPCR) showed that the relative expression levels of the three Sgt transcripts decreased by approximately 32%～60% (Sgt1), 29%～55% (Sgt2) and 25%～66% (Sgt3), respectively, while the relative expression levels of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, one of the glyphosate tolerance genes, increased by about 48%～135%. High performance liquid chromatography (HPLC) revealed that the GA content of green tissues did not show a significant difference between transgenic lines and the wild type. Importantly, the GA content in the tuber of transgenic lines decreased by 46%～59% (Zhuangshu No.3) and 42%～62% (Favorita), respectively. We propose that silencing multiple genes of the Sgt gene family reduces the accumulation of GAs in potato tubers. Hence we provide a basis to deeply understand the distribution and accumulation of GAs in different potato tissues, and may promote innovation and development for potato germplasm resources.
The formation of β-amyloid peptide (Aβ) oligomers is an important event in the pathogenesis of Alzheimer’s disease (AD). However, identification of the aggregation state of Aβ42 is essential for studies of cytotoxicity and inhibition of aggregation. A series of reasonable and simple methods were evaluated to distinguish Aβ42 monomers and oligomers based on previous studies, such as optimization of the Aβ42 amount, increase of the crosslinker, size exclusion chromatography (SEC) and thioflavin T (ThT) fluorescence analysis, etc. The following modification and optimization results are obtained: (1) Optimization of electrophoresis and electron microscopy, which are commonly used for detection of Aβ42 monomers and oligomers, has strict requirements for the amount of Aβ42. A concentration of 16.5% tris-tricine gel is best suited for electrophoretic analysis of Aβ42. For silver staining, the Aβ42 quantity in each lane is 0.5 μg. Aβ42 monomer at a concentration of 50 μg/mL is most suitable for electron microscope observation. (2) Addition of crosslinker BS3 at the final concentration of 0.3 mmol/L was found to be more effective for differentiation of Aβ42 aggregation forms. (3) Size exclusion chromatography (SEC) can be used to separate Aβ42 monomers and oligomers depending on pore size under non-denaturing conditions, but previous reports did not show the amount of Aβ42. Our methods provided detailed information for the amounts of Aβ42. The amount of Aβ42 sample added must be higher than 6.75 μg per time in SEC analysis when compared with Aβ40. (4) Cytotoxicity test and ThT analysis can be used to reveal whether a method is suitable for identifying the Aβ42 oligomerization state. Refinement and improvement of these methods will provide a reference for the study of Aβ42 in patients with Alzheimer’s disease. Some methods for the identification of specific Aβ42 oligomers have not been mentioned in this paper, and further studies are needed.
