Novel protein pp3501 mediates the inhibitory effect of sodium butyrate on SH-SY5Y cell proliferation

Sodium butyrate, a new potential therapeutic drug, improves the efficacy of chemo- and immunotherapy of cancer under unknown mechanisms. A novel gene pp3501 is significantly induced in SH-SY5Y neuroblastoma cells upon sodium butyrate treatment. Therefore, this study has cloned pp3501 cDNA by RT-PCR and generated its recombinant fusion protein and anti-serum subsequently. The pp3501 protein localized mainly in the nucleus, as detected by immunocytochemistry and the expression of pp3501-EGFP fusion protein. pp3501 inhibited the proliferation of SH-SY5Y cells, arrested the cell cycle at G1 phase, and sensitized the SH-SY5Y cells to sodium butyrate treatment. These results provide a new mechanism of sodium butyrate inhibiting cancer cell proliferation as well as a new avenue for the future research on the functions of pp3501.     

Sodium valproate potentiates staurosporine‐induced apoptosis in neuroblastoma cells via Akt/survivin independently of HDAC inhibition

Sodium valproate (VPA) has been recently identified as a selective class I histone deacetylase (HDAC) inhibitor and explored for its potential as an anti‐cancer agent. The anti‐cancer properties of VPA are generally attributed to its HDAC inhibitory activity indicating a clear overlap of these two actions, but the underlying mechanisms of its anti‐tumor effects are not clearly elucidated. The present study aimed to delineate the molecular mechanism of VPA in potentiating cytotoxic effects of anti‐cancer drugs with focus on inhibition of HDAC activity. Using human neuroblastoma cell lines, SK‐N‐MC, SH‐SY5Y, and SK‐N‐SH, we show that non‐toxic dose (2 mM) of VPA enhanced staurosporine (STS)‐induced cell death as assessed by MTT assay, PARP cleavage, hypodiploidy, and caspase 3 activity. Mechanistically, the effect of VPA was mediated by down regulation of survivin, an anti‐apoptotic protein crucial in resistance to STS‐mediated cytotoxicity, through Akt pathway. Knock down of class I HDAC isoforms remarkably inhibited HDAC activity comparable with that of VPA but had no effect on STS‐induced apoptosis. Moreover, MS‐275, a structurally distinct class I HDAC inhibitor did not affect STS‐mediated apoptosis, nor decrease the levels of survivin and Akt. Valpromide (VPM), an amide analog of VPA that does not inhibit HDAC also potentiated cell death in NB cells associated with decreased survivin and Akt levels suggesting that HDAC inhibition might not be crucial for STS‐induced apoptosis. The study provides new information on the possible molecular mechanism of VPA in apoptosis that can be explored in combination therapy in cancer. J. Cell. Biochem. 114: 854–863, 2013. © 2012 Wiley Periodicals, Inc.     

Early pathogenesis in the adult-onset neurodegenerative disease amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating paralytic disorder caused by dysfunction and degeneration of motor neurons starting in adulthood. Most of our knowledge about the pathophysiological mechanisms of ALS comes from transgenic mice models that emulate a subgroup of familial ALS cases (FALS), with mutations in the gene encoding superoxide dismutase (SOD1). In the more than 15 years since these mice were generated, a large number of abnormal cellular mechanisms underlying motor neuron degeneration have been identified, but to date this effort has led to few improvements in therapy, and no cure. Here, we consider that this surfeit of mechanisms is best interpreted by current insights that suggest a very early initiation of pathology in motor neurons, followed by a diversity of secondary cascades and compensatory mechanisms that mask symptoms for decades, until trauma and/or aging overloads their protective function. This view thus posits that adult‐onset ALS is the consequence of processes initiated during early development. In fact, motor neurons in neonatal mutant SOD mice display important alterations in their intrinsic electrical properties, synaptic inputs and morphology that are accompanied by subtle behavioral abnormalities. We consider evidence that human mutant SOD1 protein in neonatal hSOD1^G93A mice instigates motor neuron degeneration by increasing persistent sodium currents and excitability, in turn altering synaptic circuits that control excessive motor neuron firing and leads to excitotoxicity. We also discuss how therapies that are aimed at suppressing abnormal neuronal activity might effectively mitigate or prevent the onset of irreversible neuronal damage in adulthood. J. Cell. Biochem. 113: 3301–3312, 2012. © 2012 Wiley Periodicals, Inc.     

Inhibition of butyrate uptake by the primary bile salt chenodeoxycholic acid in intestinal epithelial cells

Colorectal cancer (CRC) is one of the most common cancers worldwide. Epidemiological and experimental studies suggest that bile acids may play a role in CRC etiology. Our aim was to characterize the effect of the primary bile acid chenodeoxycholic acid (CDCA) upon(14) C-BT uptake in tumoral (Caco-2) and non-tumoral (IEC-6) intestinal epithelial cell lines. A 2-day exposure to CDCA markedly and concentration-dependently inhibited (14) C-BT uptake by IEC-6 cells (IC(50) = 120 μM), and, less potently, by Caco-2 cells (IC(50) = 402 μM). The inhibitory effect of CDCA upon (14) C-BT uptake did not result from a decrease in cell proliferation or viability. In IEC-6 cells: (1) uptake of (14) C-BT involves both a high-affinity and a low-affinity transporter, and CDCA acted as a competitive inhibitor of the high-affinity transporter; (2) CDCA inhibited both Na(+)-coupled monocarboxylate cotransporter 1 (SMCT1)- and H(+)-coupled monocarboxylate transporter 1 (MCT1)-mediated uptake of (14) C-BT; (3) CDCA significantly increased the mRNA expression level of SMCT1; (4) inhibition of (14) C-BT uptake by CDCA was dependent on CaM, MAP kinase (ERK1/2 and p38 pathways), and PKC activation, and reduced by a reactive oxygen species scavenger. Finally, BT (5 mM) decreased IEC-6 cell viability and increased IEC-6 cell differentiation, and CDCA (100 μM) reduced this effect. In conclusion, CDCA is an effective inhibitor of (14) C-BT uptake in tumoral and non-tumoral intestinal epithelial cells, through inhibition of both H(+) -coupled MCT1- and SMCT1-mediated transport. Given the role played by BT in the intestine, this mechanism may contribute to the procarcinogenic effect of CDCA at this level.     

Reversal effects of pantoprazole on multidrug resistance in human gastric adenocarcinoma cells by down-regulating the V-ATPases/mTOR/HIF-1α/P-gp and MRP1 signaling pathway in vitro and in vivo

To investigate reversal effects of pantoprazole (PPZ) on multidrug resistance (MDR) in human gastric adenocarcinoma cells in vivo and in vitro. Human gastric adenocarcinoma cell SGC7901 was cultured in RPMI‐1640 medium supplemented with 10% fetal bovine serum and antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Adriamycin (ADR)‐resistant cells were cultured with addition of 0.8 µg/ml of ADR maintaining MDR phenotype. ADR was used to calculate ADR releasing index; CCK‐8 Assay was performed to evaluate the cytotoxicity of anti‐tumor drugs; BCECF‐AM pH‐sensitive fluorescent probe was used to measure intracellular pH (pHi) value of cells, whereas pH value of medium was considered as extracellular pH (pHe) value; Western blotting and immunofluorescent staining analyses were employed to determine protein expressions and intracellular distributions of vacuolar H⁺‐ATPases (V‐ATPases), mTOR, HIF‐1α, P‐glycoprotein (P‐gp), and multidrug resistant protein 1 (MRP1); SGC7901 and SGC7901/ADR cells were inoculated in athymic nude mice. Thereafter, effects of ADR with or without PPZ pretreatment were compared by determining the tumor size and weight, apoptotic cells in tumor tissues were detected by TUNEL assay. At concentrations greater than 20 µg/ml, PPZ pretreatment reduced ADR releasing index and significantly enhanced intracellular ADR concentration of SGC7901 (P < 0.01). Similarly, PPZ pretreatment significantly decreased ADR releasing index of SGC7901/ADR dose‐dependently (P < 0.01). PPZ pretreatment also decreased cell viabilities of SGG7901 and SGC7901/ADR dose‐dependently. After 24‐h PPZ pretreatment, administration of chemotherapeutic agents demonstrated maximal cytotoxic effects on SGC7901 and SGC7901/ADR cells (P < 0.05). The resistance index in PPZ pretreatment group was significantly lower than that in non‐PPZ pretreatment group (3.71 vs. 14.80). PPZ at concentration >10 µg/ml significantly decreased pHi in SGC7901 and SGC7901/ADR cells and diminished or reversed transmembrane pH gradient (P < 0.05). PPZ pretreatment also significantly inhibited protein expressions of V‐ATPases, mTOR, HIF‐1α, P‐gp, and MRP1, and alter intracellular expressions in parent and ADR‐resistant cells (P < 0.05). In vivo experiments further confirmed that PPZ pretreatment could enhance anti‐tumor effects of ADR on xenografted tumor of nude mice and also improve the apoptotic index in xenografted tumor tissues. PPZ pretreatment enhances the cytotoxic effects of anti‐tumor drugs on SGC7901 and reverse MDR of SGC7901/ADR by downregulating the V‐ATPases/mTOR/HIF‐1α/P‐gp and MRP1 signaling pathway. J. Cell. Biochem. 113: 2474–2487, 2012. © 2012 Wiley Periodicals, Inc.