Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3.

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and ß-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.

A Reporter System Evaluates Tumorigenesis, Metastasis, ß-catenin/MMP Regulation, and Druggability.

Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and Mmp9 promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of MMP9 were found in colon cancer cases. We found that proximal promoter sequences of MMP9 in murine and human contained conserved binding sites for transcription factors ß-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine Mmp9 promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by ß-catenin signaling stimulator lithium chloride. The Mmp9 promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the Mmp9 promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit Mmp9 promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the Mmp9 promoter in vitro, potentially through inhibition of ß-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as ß-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and Mmp9 promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and ß-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.

The Biological Efficacy of Natural Products against Acute and Chronic Inflammatory Diseases in the Oral Region.

The oral inflammatory diseases are divided into two types: acute and chronic inflammatory diseases. In this review, we summarize the biological efficacy of herbal medicine, natural products, and their active ingredients against acute and chronic inflammatory diseases in the oral region, especially stomatitis and periodontitis. We review the effects of herbal medicines and a biscoclaurin alkaloid preparation, cepharamthin, as a therapy against stomatitis, an acute inflammatory disease. We also summarize the effects of herbal medicines and natural products against periodontitis, a chronic inflammatory disease, and one of its clinical conditions, alveolar bone resorption. Recent studies show that several herbal medicines such as kakkonto and ninjinto reduce LPS-induced PGE 2 production by human gingival fibroblasts. Among herbs constituting these herbal medicines, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly reduce PGE 2 production. Moreover, anti-osteoclast activity has been observed in some natural products with anti-inflammatory effects used against rheumatoid arthritis such as carotenoids, flavonoids, limonoids, and polyphenols. These herbal medicines and natural products could be useful for treating oral inflammatory diseases.

Sleep bruxism frequency and platelet serotonin transporter activities in young adult subjects.

PURPOSE: To evaluate correlations between serotonin transporter (SERT) uptake ability in human peripheral platelets and sleep bruxism (SB) frequency. METHODS: Subjects were consecutively recruited from sixth-year students at Okayama University Dental School. Subjects were excluded if they (1) were receiving orthodontic treatment, (2) had a dermatological disease, (3) had taken an antidepressant within 6 months, or (4) had used an oral appliance within 6 months. SB frequency was determined as the summary score of three consecutive night assessments using a self-contained electromyography detector/analyzer in their home. Fasting peripheral venous blood samples were collected in the morning following the final SB assessment. SERT amount and platelet number were quantified via an ELISA assay and flow cytometry, respectively. Functional SERT characterization, 5-hydroxytryptamine (5-HT) uptake, maximum velocity (V max), and an affinity constant (K m ) were assessed with a [(3)H] 5-HT uptake assay. The correlations between these variables and SB level were evaluated. RESULTS: Among 50 eligible subjects (26 males, mean age 25.4 ± 2.41 years), 7 were excluded because of venipuncture failure, smoking, and alcohol intake during the experimental period. A small but significant negative correlation between SB level and [(3)H] 5-HT uptake was observed (Spearman's correlation R (2) = 0.063, p = 0.04). However, there were no significant correlations between SB level and total platelet amount, SERT, V max, and K m values (p = 0.08, 0.12, 0.71, and 0.68, respectively). CONCLUSIONS: Platelet serotonin uptake is significantly associated with SB frequency, yet only explains a small amount of SB variability.

Antiallodynic action of 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), a betaine/GABA transporter inhibitor.

The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 µM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 µM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.

Metallothionein deficiency exacerbates diabetic nephropathy in streptozotocin-induced diabetic mice.

Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in the diabetic kidney; however, the role of MT in renal function remains unclear. We therefore investigated whether MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT(-/-)) and MT(+/+) mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS), and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT(-/-) mice compared with diabetic MT(+/+) mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in the diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT(-/-) mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy.

Protective effect of cepharanthin on cisplatin-induced renal toxicity through metallothionein expression.

AIMS: Cisplatin (CDDP) is a potent anticancer agent, but severe renal toxicity can limit its use. We investigated the protective effect of cepharanthin (CE), a biscoclaurin alkaloid, on the renal toxicity of CDDP. MAIN METHODS: Mice were given CDDP along with CE. Effects of CE on CDDP toxicity were investigated by assaying markers of renal toxicity together with MT expression, and by histopathological examination of the kidney. MT-null mice were also examined. KEY FINDINGS: CE induced expression of metallothionein (MT). Pre-administration of CE attenuated an increase in blood urea nitrogen (BUN) concentrations after the CDDP injection. A histochemical analysis demonstrated protection against CDDP-induced necrocytosis of kidney tissues by CE. The protective effect of CE did not occur in the MT-null mice. SIGNIFICANCE: Pretreatment with CE may reduce the renal toxicity of CDDP through expression of MT.

High glucose increases metallothionein expression in renal proximal tubular epithelial cells.

Metallothionein (MT) is an intracellular metal-binding, cysteine-rich protein, and is a potent antioxidant that protects cells and tissues from oxidative stress. Although the major isoforms MT-1 and -2 (MT-1/-2) are highly inducible in many tissues, the distribution and role of MT-1/-2 in diabetic nephropathy are poorly understood. In this study, diabetes was induced in adult male rats by streptozotocin, and renal tissues were stained with antibodies for MT-1/-2. MT-1/-2 expression was also evaluated in mProx24 cells, a mouse renal proximal tubular epithelial cell line, stimulated with high glucose medium and pretreated with the antioxidant vitamin E. MT-1/-2 expression was gradually and dramatically increased, mainly in the proximal tubular epithelial cells and to a lesser extent in the podocytes in diabetic rats, but was hardly observed in control rats. MT-1/-2 expression was also increased by high glucose stimulation in mProx24 cells. Because the induction of MT was suppressed by pretreatment with vitamin E, the expression of MT-1/-2 is induced, at least in part, by high glucose-induced oxidative stress. These observations suggest that MT-1/-2 is induced in renal proximal tubular epithelial cells as an antioxidant to protect the kidney from oxidative stress, and may offer a novel therapeutic target against diabetic nephropathy.

Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity.

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.

Protective effects of metallothionein against dopamine quinone-induced dopaminergic neurotoxicity.

Dopamine (DA) quinone as DA neuron-specific oxidative stress conjugates with cysteine residues in functional proteins to form quinoproteins. Here, we examined the effects of cysteine-rich metal-binding proteins, metallothionein (MT)-1 and -2, on DA quinone-induced neurotoxicity. MT quenched DA semiquinones in vitro. In dopaminergic cells, DA exposure increased quinoproteins and decreased cell viability; these were ameliorated by pretreatment with MT-inducer zinc. Repeated L-DOPA administration markedly elevated striatal quinoprotein levels and reduced the DA nerve terminals specifically on the lesioned side in MT-knockout parkinsonian mice, but not in wild-type mice. Our results suggested that intrinsic MT protects against L-DOPA-induced DA quinone neurotoxicity in parkinsonian mice by its quinone-quenching property.

Expression pattern of cisplatin-induced metallothionein isoforms in squamous cell carcinoma.

Cisplatin (CDDP) is a useful drug for the treatment of malignant solid tumors of the head and neck. Because CDDP includes the heavy metal platinum as a component, it is thought metallothionein (MT) may be involved in CDDP-resistance. However, functional differences between the four MT isoforms (MT-I, II, III and IV) remain unclear. The aim of this study was to investigate the relationship between MT isoform expression and CDDP-resistance. Two human tongue squamous cell carcinoma cell lines not exposed to anticancer chemotherapy were studied. The cell lines were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis before and after CDDP-treatment. Both cell lines expressed MT-I/II and MT-IV isoforms but not the MT-III isoform. Following CDDP treatment, MT-I/II mRNA levels were induced only in the CDDP-resistant cell line. Our results showed that expression of the MT I/II isoform was induced by CDDP treatment, and may play an important role in CDDP-resistance in squamous cell carcinoma of the human tongue.