Combination of Evodiamine with Berberine Reveals a Regulatory Effect on the Phenotypic Transition of Colon Epithelial Cells Induced by CCD-18Co.

Objective Transdifferentiation exists between stromal cells or between stromal cells and cancer cells. Evodiamine and berberine are predominant pharmacological components of Zuojin pill, a prescription of Traditional Chinese Medicine, playing crucial functions in remolding of tumor microenvironment. This study aimed to explore the effect of combination of evodiamine with berberine (cBerEvo) on the phenotypic transition of colon epithelial cells induced by tumor-associated fibroblasts, as well as the involved mechanisms.Methods Human normal colon epithelial cell line HCoEpiC cells were treated with the prepared conditioned medium of CCD-18Co, a human colon myofibroblast line, to induce epithelial-mesenchymal transition. Phase contrast microscope was used to observe the morphological changes. Epithelial-mesenchymal transition markers including E-cadherin, vimentin and alpha-smooth muscle actin (α-SMA) were observed with immunofluorescence microscopy. Migration was assessed by wound healing assay. Western blotting was used to detect the expressions of E-cadherin, vimentin, α-SMA, Snail, ZEB1 and Smads. Results In contrast to the control, the tumor-associated fibroblasts-like CCD-18Co cells induced down-regulation of E-cadherin and up-regulation of vimentin, α-SMA, Snail and ZEB1 (P<0.05), and promoted migration of HCoEpiCs (P<0.05), with over expression of Smads including Smad2, p-Smad2, Smad3, p-Smad3 and Smad4 (P<0.05), which were abolished by a transforming growth factor-ß (TGF-ß) receptor inhibitor LY364947 and by cBerEvo in a concentration dependent manner. In addition, cBerEvo-inhibited ratios of p-Smad2/Smad2 and p-Smad3/Smad3 were also dose dependent.Conclusion The above results suggest that cBerEvo can regulate the differentiation of colon epithelial cells induced by CCD-18Co through suppressing activity of TGF-ß/Smads signaling pathway.

Berberine reversed the epithelial-mesenchymal transition of normal colonic epithelial cells induced by SW480 cells through regulating the important components in the TGF-ß pathway.

Stroma-tumor interactions within microenvironment play a crucial role in tumor development and growth. Cellular transdifferentiation in the stroma is a prerequisite for tumor formation. Targeting the interactions maybe a promising anticancer strategy. Berberine (BBR) has been confirmed to have anticancer and anti-inflammatory effects. We found for the first time that colon cancer cells SW480 induced spindle-like morphological changes and downregulation of E-cadherin and upregulation of vimentin and alpha-smooth muscle actin in colon epithelial cells HCoEpiCs by using transwell coculture system and conditioned medium from SW480. The conditioned medium also promoted the migration of HCoEpiCs. This transition was inhibited by a transforming growth factor-ß receptor inhibitor LY364947. BBR (50 and 100 µg/ml) reversed the EMT-like transition and repressed the migration in HCoEpiCs. Further results demonstrated that downregulation of TßRII, Smad2, p-Smad3, and overexpression of Smad3 participated in the SW480-induced phenotypic transition of HCoEpiCs. In addition, BBR upregulated the expressions of TßRII, Smad2, and p-Smad3. In conclusion, our findings suggest that BBR exerts the anti-EMT and antimigration effect by mediating the expression of TßRII, Smad2, and p-Smad3.

Difference of TGF-ß/Smads signaling pathway in epithelial-mesenchymal transition of normal colonic epithelial cells induced by tumor-associated fibroblasts and colon cancer cells.

Tumor microenvironment (TME) crucially functions in tumor initiation and progression. Stroma-tumor interactions and cellular transdifferentiation are the prerequisite for tumor formation. Transforming growth factor-ß (TGF-ß), a major cytokine secreted by tumor-associated fibroblasts (TAFs) and cancer cells, is a crucial player involving cell transdifferentiation. Therefore, we hypothesized that these TAFs and cancer cells also affect normal colon epithelium. In our study, we found for the first time that colon cancer cells HCT116 and TAF-like CCD-18Co cells induced epithelial-mesenchymal transition (EMT)-like transdifferentiation in colon epithelial cells HCoEpiCs, with enhanced migratio. Dysfunction of TGF-ß/Smads signal was also observed in the EMT-transformed HCoEpiCs. We wondered whether these phenomena were regulated by TGF-ß/Smads signaling pathway. A TGFß receptor kinase I (TßRI) inhibitor LY364947 was used. We found that the EMT induced by the HCT116- and CCD-18Co-derived CM was suppressed by the LY364947. Besides, different expression profiles for the components of TGF-ß/Smads pathway were found in the EMT-like HCoEpiCs, but high expression of p-Smad2/3 and Smad4 was the common feature. Our observations suggest that the mechanisms of phenotypic transition of colon epithelial cells are cellular environment-dependent, which maybe a basis of potential therapy targeting TME.

Synaptic released zinc promotes tau hyperphosphorylation by inhibition of protein phosphatase 2A (PP2A).

Hyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer disease (AD), and the tangle distribution largely overlaps with zinc-containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pretreatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser-396, Ser-404, Thr-231, and Thr-205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus were detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase 2A (PP2A), and this inactivation can be reversed by pretreatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition.

Berberine inhibits epithelial-mesenchymal transition and promotes apoptosis of tumour-associated fibroblast-induced colonic epithelial cells through regulation of TGF-ß signalling.

Tumour-associated fibroblasts (TAFs) mediate the differentiation of adjacent stromal cells. Berberine (BBR), a monomer of traditional Chinese herbs, exhibits a potent therapeutic effect against cancer. However, the effects of BBR on the differentiation of normal colonic epithelial cells induced by TAFs have not been determined. In the present study, we selected the TAF-like myofibroblast cell line CCD-18Co. CCD-18Co-derived conditioned medium (CM) and co-culture induced epithelial-mesenchymal transition (EMT) changes in colonic epithelial HCoEpiC cells with decreased E-cadherin and increased vimentin and α-SMA expression. In addition, CCD-18Co stimulated the expression of ZEB1 and Snail and promoted motility. We used LY364947, a TGF-ß receptor kinase type I (TßRI) inhibitor, and BBR. Our results showed that LY364947 and BBR inhibited these phenomena. BBR decreased the expression of ZEB1 and Snail, and this effect was concentration dependent. BBR also downregulated the expression of TßRI, TßRII, Smad2/p-Smad2 and Smad3/p-Smad3. In addition, BBR induced apoptosis in EMT-like HCoEpiC cells in a concentration-dependent manner with upregulation of Bax and downregulation of Bcl-2. However, VX-702, an inhibitor of p38 MAPK, significantly suppressed the apoptosis rate. BBR promoted the expression of p38 MAPK and phosphorylated p38 MAPK. In conclusion, berberine inhibits EMT and promotes apoptosis in TAF-induced colonic epithelial cells through mediation of the Smad-dependent and SMAD-independent TGF-ß signalling pathways.

Trichostatin a inhibits phenotypic transition and induces apoptosis of the TAF-treated normal colonic epithelial cells through regulation of TGF-ß pathway.

Tumor-associated fibroblasts (TAFs) contribute to transdifferentiation of stromal cells in tumor microenvironment. Epithelial-mesenchymal transition (EMT) is a procedure of phenotypic remodeling of epithelial cells and extensively exists in local tumoral stroma. Histone deacetylase (HDAC) inhibitor Tricostatin A (TSA) and sodium butyrate (SB) are reported to play important roles in the regulation of biological behaviour of cancer cells. However, whether TSA or SB is involved in control of EMT in colon epithelial cells induced by TAFs remains unidentified. In present study, we used conditioned medium (CM) form TAF-like CCD-18Co cells to stimulate 2D- and 3D-cultured colon epithelial HCoEpiC cells for 24 h and 4 d. We found that the CCD-18Co CM triggered multiple morphological changes in HCoEpiCs including prolonged cell diameters, down-regulation of E-cadherin and up-regulation of vimentin and α-SMA. Besides, ZEB1 and Snail expression and migration were also promoted by the CM. These phenomena were abolised by 5 µg/ml LY364947, a TGF-ß receptor inhibitor. CCD-18Co induced up-regulation of HDAC1 and HDAC2 in the 2D and 3D models, while no change of HDAC4 exprerssion was found. Treatment of 2 µg/ml TSA reversed the CCD-18Co-induced morphological changes and migration of the HCoEpiCs, and suppressed the downregulation of E-cadherin and upregulation of vimentin, α-SMA, ZEB1 and Snail. However, the suppressive effect of 4 mg/ml SB on the EMT was not observed. TSA down-regulated the expressions of Smad2/3, p-Smad2/3 amd HDAC4. Besides, TSA promoted the apoptosis rate (36.84 ± 6.52%) comparing with the CCD-18Co-treated HCoEpiCs (3.52 ± 0.85%, P < 0.05), with promotion of Bax (0.5893±0.0498 in 2D and 0.8867±0.0916 in 3D) and reduction of Bcl-2 (0.0476±0.0053 in 2D and 0.0294±0.0075 in 3D). TSA stimulated expression of phosphorylated-p38 MAPK in 2D (0.3472±0.0249) and 3D (0.3188±0.0248). After pre-treatment with p38 MAPK inhibitor VX-702 (0.5 mg/ml), the apoptosis rate of TSA was decreased in 2D (10.32%) and 3D (5.26%). Our observations demonstrate that epigenetic treatment with HDAC inhibitor TSA may be a useful therapeutic tool for the reversion of TAF-induced EMT in colon epithelium through mediating canonical Smads pathway and non-canonical p38 MAPK signalling.

CIP2A Causes Tau/APP Phosphorylation, Synaptopathy, and Memory Deficits in Alzheimer's Disease.

Protein phosphatase 2A (PP2A) inhibition causes hyperphosphorylation of tau and APP in Alzheimer's disease (AD). However, the mechanisms underlying the downregulation of PP2A activity in AD brain remain unclear. We demonstrate that Cancerous Inhibitor of PP2A (CIP2A), an endogenous PP2A inhibitor, is overexpressed in AD brain. CIP2A-mediated PP2A inhibition drives tau/APP hyperphosphorylation and increases APP ß-cleavage and Aß production. Increase in CIP2A expression also leads to tau mislocalization to dendrites and spines and synaptic degeneration. In mice, injection of AAV-CIP2A to hippocampus induced AD-like cognitive deficits and impairments in long-term potentiation (LTP) and exacerbated AD pathologies in neurons. Indicative of disease exacerbating the feedback loop, we found that increased CIP2A expression and PP2A inhibition in AD brains result from increased Aß production. In summary, we show that CIP2A overexpression causes PP2A inhibition and AD-related cellular pathology and cognitive deficits, pointing to CIP2A as a potential target for AD therapy.

Extrasynaptic NMDA receptor-induced tau overexpression mediates neuronal death through suppressing survival signaling ERK phosphorylation.

Intracellular accumulation of the hyperphosphorylated tau is a pathological hallmark in the brain of Alzheimer disease. Activation of extrasynaptic NMDA receptors (E-NMDARs) induces excitatory toxicity that is involved in Alzheimer's neurodegeneration. However, the intrinsic link between E-NMDARs and the tau-induced neuronal damage remains elusive. In the present study, we showed in cultured primary cortical neurons that activation of E-NMDA receptors but not synaptic NMDA receptors dramatically increased tau mRNA and protein levels, with a simultaneous neuronal degeneration and decreased neuronal survival. Memantine, a selective antagonist of E-NMDARs, reversed E-NMDARs-induced tau overexpression. Activation of E-NMDARs in wild-type mouse brains resulted in neuron loss in hippocampus, whereas tau deletion in neuronal cultures and in the mouse brains rescued the E-NMDARs-induced neuronal death and degeneration. The E-NMDARs-induced tau overexpression was correlated with a reduced ERK phosphorylation, whereas the increased MEK activity, decreased binding and activity of ERK phosphatase to ERK, and increased ERK phosphorylation were observed in tau knockout mice. On the contrary, addition of tau proteins promoted ERK dephosphorylation in vitro. Taking together, these results indicate that tau overexpression mediates the excitatory toxicity induced by E-NMDAR activation through inhibiting ERK phosphorylation.

Zinc binds to and directly inhibits protein phosphatase 2A in vitro.

Zinc induces protein phosphatase 2A (PP2A) inactivation and tau hyperphosphorylation through PP2A (tyrosine 307) phosphorylation in cells and the brain, but whether Zn(2+) has a direct inhibitory effect on PP2A is not clear. Here we explored the effect of Zn(2+) on PP2A and their direct interaction in vitro. The results showed that Zn(2+) mimicked the inhibitory effect of okadaic acid on protein phosphatase and prevented tau dephosphorylation in N2a cell lysates. PP2A activity assays indicated that a low concentration (10 µmol/L) of Zn(2+) inhibited PP2A directly. Further Zn(2+)-IDA-agarose affinity binding assays showed that Zn(2+) bound to and inhibited PP2Ac(51-270) but not PP2Ac(1-50) or PP2Ac(271-309). Taken together, Zn(2+) inhibits PP2A directly through binding to PP2Ac(51-270) in vitro.

Helicobacter pylori filtrate induces Alzheimer-like tau hyperphosphorylation by activating glycogen synthase kinase-3ß.

Abnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Helicobacter pylori (H. pylori) infection has been reported to be related with a high risk of AD, but the direct laboratory evidence is lacking. Here we explored the effect of H. pylori infection on tau phosphorylation. The results showed that H. pylori filtrate induced significant tau hyperphosphorylation at several AD-related tau phosphorylation sites, such as Thr205, Thr231, and Ser404, both in mouse neuroblastoma N2a cells and rat brains with activation of glycogen synthase kinase-3ß (GSK-3ß). Application of GSK-3 inhibitors efficiently attenuated the H. pylori-induced tau hyperphosphorylation. Our data provide evidence supporting the role of H. pylori infection in AD-like tau pathology, suggesting that H. pylori eradication may be beneficial in the prevention of tauopathy.

A novel tacrine-dihydropyridine hybrid (-)SCR1693 induces tau dephosphorylation and inhibits Aß generation in cells.

AChE inhibitors are the first choice for the treatment of Alzheimer׳s disease (AD), but they could only delay the progression of cognitive and behavioral dysfunction, and fail to reverse neuronal damage. Calcium channel blockers have been identified to have protective effect on neurons. Thus, therapy targeting both AChE and calcium channels is supposed to be more effective in AD treatment. In the present study, we explored the effect of a synthesized juxtaposition of an AChE inhibitor and a Calcium channel blocker (named (-)SCR1693) on tau phosphophorylation and Aß generation. The results showed that: (1) Compared with higher concentrations, (-)SCR1693 incubation in low concentrations such as 0.4, 2, 4µM for 24h did not affect the cell viability of HEK293/tau (HEK293 cells stably transfected with human tau40) and N2a/APP (N2a cells stably transfected with human APP) cells; (2) long-term treatment of cells with (-)SCR1693 (0.4, 2, 5µM) (24h) induced tau dephosphorylation and reduced the total tau level in HEK293/tau cells. Short-term treatment (6h) also resulted in tau dephosphorylation, but did not reduce the total tau level; and (3) (-)SCR1693 (0.4, 2, 4µM) incubation inhibited Aß generation and release dramatically in N2a/APP cells. We conclude that the novel tacrine-dihydropyridine hybrid (-)SCR1693 in low concentrations could reduce total and phosphorylated tau levels, inhibit the generation and release of Aß in cells. Thus, (-)SCR1693 may be a potential candidate for effectively treating AD.

Helicobacter pylori filtrate impairs spatial learning and memory in rats and increases ß-amyloid by enhancing expression of presenilin-2.

Helicobacter pylori (H. pylori) infection is related with a high risk of Alzheimer's disease (AD), but the intrinsic link between H. pylori infection and AD development is still missing. In the present study, we explored the effect of H. pylori infection on cognitive function and ß-amyloid production in rats. We found that intraperitoneal injection of H. pylori filtrate induced spatial learning and memory deficit in rats with a simultaneous retarded dendritic spine maturation in hippocampus. Injection of H. pylori filtrate significantly increased Aß42 both in the hippocampus and cortex, together with an increased level of presenilin-2 (PS-2), one key component of γ-secretase involved in Aß production. Incubation of H. pylori filtrate with N2a cells which over-express amyloid precursor protein (APP) also resulted in increased PS-2 expression and Aß42 overproduction. Injection of Escherichia coli (E.coli) filtrate, another common intestinal bacterium, had no effect on cognitive function in rats and Aß production in rats and cells. These data suggest a specific effect of H. pylori on cognition and Aß production. We conclude that soluble surface fractions of H. pylori may promote Aß42 formation by enhancing the activity of γ-secretase, thus induce cognitive impairment through interrupting the synaptic function.

High dose zinc supplementation induces hippocampal zinc deficiency and memory impairment with inhibition of BDNF signaling.

Zinc ions highly concentrate in hippocampus and play a key role in modulating spatial learning and memory. At a time when dietary fortification and supplementation of zinc have increased the zinc consuming level especially in the youth, the toxicity of zinc overdose on brain function was underestimated. In the present study, weaning ICR mice were given water supplemented with 15 ppm Zn (low dose), 60 ppm Zn (high dose) or normal lab water for 3 months, the behavior and brain zinc homeostasis were tested. Mice fed high dose of zinc showed hippocampus-dependent memory impairment. Unexpectedly, zinc deficiency, but not zinc overload was observed in hippocampus, especially in the mossy fiber-CA3 pyramid synapse. The expression levels of learning and memory related receptors and synaptic proteins such as NMDA-NR2A, NR2B, AMPA-GluR1, PSD-93 and PSD-95 were significantly decreased in hippocampus, with significant loss of dendritic spines. In keeping with these findings, high dose intake of zinc resulted in decreased hippocampal BDNF level and TrkB neurotrophic signaling. At last, increasing the brain zinc level directly by brain zinc injection induced BDNF expression, which was reversed by zinc chelating in vivo. These results indicate that zinc plays an important role in hippocampus-dependent learning and memory and BDNF expression, high dose supplementation of zinc induces specific zinc deficiency in hippocampus, which further impair learning and memory due to decreased availability of synaptic zinc and BDNF deficit.

Zinc induces protein phosphatase 2A inactivation and tau hyperphosphorylation through Src dependent PP2A (tyrosine 307) phosphorylation.

The activity of protein phosphatase (PP) 2A is downregulated and promotes the hyperphosphorylation of tau in the brains of Alzheimer's disease (AD), but the mechanism for PP2A inactivation has not been elucidated. We have reported that PP2A phosphorylation at tyrosine 307 (Y307) is involved in PP2A inactivation. Here, we further studied the upstream mechanisms for PP2A phosphorylation and inactivation. We found that zinc, a heavy metal ion that is widely distributed in the normal brain and accumulated in the susceptible regions of AD brain, could induce PP2A inhibition, phosphorylation of PP2A at Y307 and tau hyperphosphorylation both in rat brains and cultured N2a cells, while zinc chelating prevented these changes completely. Upregulation of PP2A chemically or genetically attenuated zinc-induced tau hyperphosphorylation, whereas mutation of Y307 to phenylalanine abolished the zinc-induced tyrosine phosphorylation and inactivation of PP2A. Zinc could activate Src, while PP2, a specific Src family kinases inhibitor, attenuated zinc-induced PP2A phosphorylation and inactivation, indicating that zinc induces PP2A Y307 phosphorylation and inactivation through Src activation. In human tau transgenic mice, zinc chelator rescued PP2A activity, prevented Src activation, and reduced hyperphosphorylated and insoluble tau levels. We concluded that zinc induces PP2A inactivation and tau hyperphosphorylation through Src-dependent pathway, regulation of zinc homeostasis may be a promising therapeutic for AD and the related tauopathies.