GSH-dependent antioxidant defense contributes to the acclimation of colon cancer cells to acidic microenvironment

<p>Due to increased glycolysis and poor local perfusion, solid tumors are usually immersed in an acidic microenvironment. While extracellular acidosis is cytotoxic, cancer cells eventually become acclimated to it. While previous studies have addressed the acute effect of acidosis on cancer cells, little is known about how cancer cells survive chronic acidosis. In this study we exposed colorectal cancer (CRC) cells (HCT15, HCT116 and LoVo) to acidic pH (pH 6.5) continuously for over three months and obtained CRC cells that become acclimated to acidic pH, designated as CRC-acidosis-acclimated or CRC-AA. We unexpectedly found that while acute exposure to low pH resulted in an increase in the level of intracellular reactive oxygen species (ROS), CRC-AA cells exhibited a significantly reduced level of ROS when compared to ancestor cells. CRC-AA cells were found to maintain a higher level of reduced glutathione, via the upregulation of CD44 and glutathione reductase (GSR), among others, than their ancestor cells. Importantly, CRC-AA cells were more sensitive to agents that deplete GSH. Moreover, downregulation of GSR by RNA interference was more deleterious to CRC-AA cells than to control cells. Together, our results demonstrate a critical role of glutathione-dependent antioxidant defense in acclimation of CRC cells to acidic extracellular pH.</p

Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

<div><p>Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.</p> </div

Figure S1 from Berberine Induces Senescence of Human Glioblastoma Cells by Downregulating the EGFR–MEK–ERK Signaling Pathway

Figure S1. DNA damage response is not required for berberine-induced senescence</p

Figure S2 from Berberine Induces Senescence of Human Glioblastoma Cells by Downregulating the EGFR–MEK–ERK Signaling Pathway

Figure S2. Downregulation of EGFR by berberine is not caused by reduced transcription</p

Immunoblots and relative mRNA levels of VCP and linkers between UPS and autophagy in control and GNE myopathy muscle biopsies.

<p>The densitometry graphs are representative of only the one chosen in the corresponding blots. A: Immunoblots of muscle homogenates of normal control and GNE myopathy muscle biopsies demonstrate in GNE myopathy a much stronger expression of HDAC6, p62, NBR1 and VCP as compared to control muscle biopsies. C = control tissue, n = 2 (Samples are from control-4 and -5); GNE myopathy, n = 5 (Samples are from HIBM-4, -5, -6, -7 and -8). B: Densitometric analysis of the blots in A performed using Quantity One. Protein loading was evaluated by the GAPDH and actin band. Data are indicated as mean±SD. Significance was determined by one-tailed unpaired t-test. The level of significance was set at P<0.05. B: Relative mRNA levels of VCP and linkers between UPS and autophagy in normal and GNE myopathy patient biopsies. A total of four samples for each group were used, and each sample was run in triplicate for real-time PCR. C = control tissue, n = 4 (Samples are from control-1, -3, -4 and -5); GNE myopathy, n = 4 (Samples are from HIBM-5, -6, -7 and -8). Relative mRNA levels of VCP and linkers were increased in GNE myopathy, VCP to 215.2%, HDAC to 135.4%, NBR1 to 95.1% and p62 to 64.5%. Data are shown as means ± SD. The level of significance was set at P<0.05. The densitometry graphs are representative of only the one chosen in the corresponding blot.</p

Figure S3 from Berberine Induces Senescence of Human Glioblastoma Cells by Downregulating the EGFR–MEK–ERK Signaling Pathway

Figure S3. EGFR inhibitor fails to induce apoptosis in U87 cells</p

Supplementary Figure Legend from Berberine Induces Senescence of Human Glioblastoma Cells by Downregulating the EGFR–MEK–ERK Signaling Pathway

Legend to supplementary figures</p

Immunoblots of AβPP and p-tau in control and GNE myopathy muscle biopsies.

<p>A: Immunoblots of muscle homogenates of two normal control and five GNE myopathy muscle biopsies demonstrate in GNE myopathy a much stronger expression of AβPP and p-tau as compared to control muscle biopsies. C = control tissue, n = 2 (Samples are from control-1 and control-2); GNE myopathy, n = 5 (Samples are from GNE myopathy -1, -2, -3, -4, and -5). B: Densitometric analysis of the blots in A performed using Quantity One. Protein loading was evaluated by the actin band. Data are indicated as mean±SD. Significance was determined by one-tailed unpaired t-test. The level of significance was set at P<0.05. The densitometry graphs are representative of only the one chosen in the corresponding blots.</p

Hydroxyurea, aphidicolin and thymidine all cause S phase arrest.

<p>Cells were treated with different drugs for 24 h in all the panels. The distribution of cell cycle was assessed by flow cytometry. The experiments were performed in triplicate. Error bars represent standard deviation. *<i>P</i><0.05, #<i>P</i><0.01, when compared with control group.</p

Immunoblots and relative mRNA levels of of endoplasmic reticulum molecular chaperones in normal control and GNE myopathy muscle biopsies.

<p>A: Immunoblots of muscle homogenates of two normal control and five GNE myopathy muscle biopsies demonstrate in GNE myopathy a much stronger expression of GRP94, GRP78, calnexin and calreticulin except for ERp72 as compared to control muscle biopsies. C = control tissue, n = 2 (Samples are from control-1 and -2); GNE myopathy, n = 5 (Samples are from HIBM2-1, -2, -3, -4, and -5) B: Densitometric analysis of the blots in A performed using Quantity One. Protein loading was evaluated by the actin band. Data are indicated as mean±SD. Significance was determined by one-tailed unpaired t-test. The level of significance was set at P<0.05.C: Relative mRNA levels of molecular chaperones in normal and GNE myopathy patient biopsies. A total of four samples for each group were used, and each sample was run in triplicate for real-time PCR. C = control tissue, n = 4 (Samples are from control-1, -3, -4 and -5); GNE myopathy, n = 4 (Samples are from HIBM-5, -6, -7 and -8). Relative mRNA levels of four molecular chaperones were significantly increased in GNE myopathy muscle biopsies, GRP94 to112.3%, GRP78 to78.1%, calnexin to165.5%, and calreticulin to 189.6%. Data are shown as means ± SD. The level of significance was set at P<0.05. The densitometry graphs are representative of only the one chosen in the corresponding blots.</p