GIRK2 potassium channels expressed by the AgRP neurons decrease adiposity and body weight in mice.

It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K+ (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.

Hypothalamic GABRA5-positive neurons control obesity via astrocytic GABA.

The lateral hypothalamic area (LHA) regulates food intake and energy balance. Although LHA neurons innervate adipose tissues, the identity of neurons that regulate fat is undefined. Here we show that GABRA5-positive neurons in LHA (GABRA5LHA) polysynaptically project to brown and white adipose tissues in the periphery. GABRA5LHA are a distinct subpopulation of GABAergic neurons and show decreased pacemaker firing in diet-induced obesity mouse models in males. Chemogenetic inhibition of GABRA5LHA suppresses fat thermogenesis and increases weight gain, whereas gene silencing of GABRA5 in LHA decreases weight gain. In the diet-induced obesity mouse model, GABRA5LHA are tonically inhibited by nearby reactive astrocytes releasing GABA, which is synthesized by monoamine oxidase B (Maob). Gene silencing of astrocytic Maob in LHA facilitates fat thermogenesis and reduces weight gain significantly without affecting food intake, which is recapitulated by administration of a Maob inhibitor, KDS2010. We propose that firing of GABRA5LHA suppresses fat accumulation and selective inhibition of astrocytic GABA is a molecular target for treating obesity.

COVID-19 spike polypeptide vaccine reduces the pathogenesis and viral infection in a mouse model of SARS-CoV-2.

The SARS-CoV-2 coronavirus, which causes a respiratory disease called COVID-19, has been declared a pandemic by the World Health Organization (WHO) and is still ongoing. Vaccination is the most important strategy to end the pandemic. Several vaccines have been approved, as evidenced by the ongoing global pandemic, but the pandemic is far from over and no fully effective vaccine is yet available. One of the most critical steps in vaccine development is the selection of appropriate antigens and their proper introduction into the immune system. Therefore, in this study, we developed and evaluated two proposed vaccines composed of single and multiple SARS-CoV-2 polypeptides derived from the spike protein, namely, vaccine A and vaccine B, respectively. The polypeptides were validated by the sera of COVID-19-vaccinated individuals and/or naturally infected COVID-19 patients to shortlist the starting pool of antigens followed by in vivo vaccination to hACE2 transgenic mice. The spike multiple polypeptide vaccine (vaccine B) was more potent to reduce the pathogenesis of organs, resulting in higher protection against the SARS-CoV-2 infection.

Short-term carcinogenicity study of N-methyl-N-nitrosourea in FVB-Trp53 heterozygous mice.

Carcinogenicity tests predict the tumorigenic potential of various substances in the human body by studying tumor induction in experimental animals. There is a need for studies that explore the use of FVB/N-Trp53em2Hwl/Korl (FVB-Trp53+/-) mice, created by TALEN-mediated gene targeting in Korea, in carcinogenicity tests. This study was performed to determine whether FVB-Trp53+/- mice are a suitable model for short-term carcinogenicity studies. To compare the carcinogenicity at different concentrations, 25, 50, and 75 mg/kg of N-methyl-N-nitrosourea (MNU), a known carcinogen, were administered intraperitoneally to FVB-Trp53+/- and wild-type male mice. After 26 weeks, the survival rate was significantly reduced in FVB-Trp53+/- mice compared to the wild-type mice in the 50 and 75 mg/kg groups. The incidence of thymic malignant lymphoma (TML) in the 50 and 75 mg/kg groups was 54.2 and 59.1% in FVB-Trp53+/- male mice, respectively. TML metastasized to the lungs, spleen, lymph nodes, liver, kidney, and heart in FVB-Trp53+/- male mice. Furthermore, the incidence of primary lung tumors, such as adenomas and adenocarcinomas, was 65.4, 62.5, and 45.4% in the FVB-Trp53+/- mice of the 25, 50, and 75 mg/kg groups, respectively. The main tumor types in FVB-Trp53+/- mice were TML and primary lung tumors, regardless of the dose of MNU administered. These results suggest that systemic tumors may result from malfunctions in the p53 gene and pathway, which is an important factor in the pathogenesis of human cancers. Therefore, FVB-Trp53 heterozygous mice are suitable for short-term carcinogenicity tests using positive carcinogens, and that the best result using MNU, a positive carcinogen, might have a single dose of 50 mg/kg.

Chrysin Induces Apoptosis and Autophagy in Human Melanoma Cells via the mTOR/S6K Pathway.

Chrysin is known to exert anti-inflammatory, antioxidant, and anticancer effects. The aim of this study was to investigate the anticancer effects of chrysin in the human melanoma cells A375SM and A375P. The results obtained demonstrated successful inhibition of the viability of these cells by inducing apoptosis and autophagy. This was confirmed by the level of apoptosis-related proteins: Bax and cleaved poly (ADP-ribose) polymerase both increased, and Bcl-2 decreased. Moreover, levels of LC3 and Beclin 1, both autophagy-related proteins, increased in chrysin-treated cells. Autophagic vacuoles and acidic vesicular organelles were observed in both cell lines treated with chrysin. Both cell lines showed different tendencies during chrysin-induced autophagy inhibition, indicating that autophagy has different effects depending on the cell type. In A375SM, the early autophagy inhibitor 3-methyladenine (3-MA) was unaffected; however, cell viability decreased when treated with the late autophagy inhibitor hydroxychloroquine (HCQ). In contrast, HCQ was unaffected in A375P; however, cell viability increased when treated with 3-MA. Chrysin also decreased the phosphorylation of mTOR/S6K pathway proteins, indicating that this pathway is involved in chrysin-induced apoptosis and autophagy for A375SM and A375P. However, studies to elucidate the mechanisms of autophagy and the action of chrysin in vivo are still needed.

Lomitapide, a cholesterol-lowering drug, is an anticancer agent that induces autophagic cell death via inhibiting mTOR.

Autophagy is a biological process that maintains cellular homeostasis and regulates the internal cellular environment. Hyperactivating autophagy to trigger cell death has been a suggested therapeutic strategy for cancer treatment. Mechanistic target of rapamycin (mTOR) is a crucial protein kinase that regulates autophagy; therefore, using a structure-based virtual screen analysis, we identified lomitapide, a cholesterol-lowering drug, as a potential mTOR complex 1 (mTORC1) inhibitor. Our results showed that lomitapide directly inhibits mTORC1 in vitro and induces autophagy-dependent cancer cell death by decreasing mTOR signaling, thereby inhibiting the downstream events associated with increased LC3 conversion in various cancer cells (e.g., HCT116 colorectal cancer cells) and tumor xenografts. Lomitapide also significantly suppresses the growth and viability along with elevated autophagy in patient-derived colorectal cancer organoids. Furthermore, a combination of lomitapide and immune checkpoint blocking antibodies synergistically inhibits tumor growth in murine MC38 or B16-F10 preclinical syngeneic tumor models. These results elucidate the direct, tumor-relevant immune-potentiating benefits of mTORC1 inhibition by lomitapide, which complement the current immune checkpoint blockade. This study highlights the potential repurposing of lomitapide as a new therapeutic option for cancer treatment.

Development of transgenic models susceptible and resistant to SARS-CoV-2 infection in FVB background mice.

Coronavirus disease (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is currently spreading globally. To overcome the COVID-19 pandemic, preclinical evaluations of vaccines and therapeutics using K18-hACE2 and CAG-hACE2 transgenic mice are ongoing. However, a comparative study on SARS-CoV-2 infection between K18-hACE2 and CAG-hACE2 mice has not been published. In this study, we compared the susceptibility and resistance to SARS-CoV-2 infection between two strains of transgenic mice, which were generated in FVB background mice. K18-hACE2 mice exhibited severe weight loss with definitive lethality, but CAG-hACE2 mice survived; and differences were observed in the lung, spleen, cerebrum, cerebellum, and small intestine. A higher viral titer was detected in the lungs, cerebrums, and cerebellums of K18-hACE2 mice than in the lungs of CAG-hACE2 mice. Severe pneumonia was observed in histopathological findings in K18-hACE2, and mild pneumonia was observed in CAG-hACE2. Atrophy of the splenic white pulp and reduction of spleen weight was observed, and hyperplasia of goblet cells with villi atrophy of the small intestine was observed in K18-hACE2 mice compared to CAG-hACE2 mice. These results indicate that K18-hACE2 mice are relatively susceptible to SARS-CoV-2 and that CAG-hACE2 mice are resistant to SARS-CoV-2. Based on these lineage-specific sensitivities, we suggest that K18-hACE2 mouse is suitable for highly susceptible model of SARS-CoV-2, and CAG-hACE2 mouse is suitable for mild susceptible model of SARS-CoV-2 infection.

Gαi/o-coupled Htr2c in the paraventricular nucleus of the hypothalamus antagonizes the anorectic effect of serotonin agents.

The anorexigenic effect of serotonergic compounds has largely been attributed to activation of serotonin 2C receptors (Htr2cs). Using mouse genetic models in which Htr2c can be selectively deleted or restored (in Htr2c-null mice), we investigate the role of Htr2c in forebrain Sim1 neurons. Unexpectedly, we find that Htr2c acts in these neurons to promote food intake and counteract the anorectic effect of serotonergic appetite suppressants. Furthermore, Htr2c marks a subset of Sim1 neurons in the paraventricular nucleus of the hypothalamus (PVH). Chemogenetic activation of these neurons in adult mice suppresses hunger, whereas their silencing promotes feeding. In support of an orexigenic role of PVH Htr2c, whole-cell patch-clamp experiments demonstrate that activation of Htr2c inhibits PVH neurons. Intriguingly, this inhibition is due to Gαi/o-dependent activation of ATP-sensitive K+ conductance, a mechanism of action not identified previously in the mammalian nervous system.

Therapeutic efficacy of new botulinum toxin identified in CCUG 7968 strain.

Botulinum neurotoxin type A (BoNT/A) induces muscle atrophy by cleaving synaptosomal-associated protein 25. Thus, BoNT/A has been actively utilized for the treatment of masseter and gastrocnemius hypertrophy. In this study, INI101 toxin was newly identified from the CCUG 7968 strain, and its therapeutic efficacy was evaluated both in vitro and in vivo. The INI101 toxin showed identical genetic sequence, amino acid sequence, and protein subunit composition to BoNT/A produced from strain Hall A. Electromyography (EMG), and immunofluorescence staining demonstrated that INI101 (at 2 ~ 8 U/rat) effectively blocked the neuromuscular junction with no toxicity in a rat model. The EMG results showed INI101 toxin-induced weight loss and volume reduction of the gastrocnemius, similar to the effects of Botox® (BTX). Histological and immunofluorescence staining was consistent with this EMG result, showing that INI101 toxin caused muscle fiber reduction in the gastrocnemius. Notably, INI101 toxin diffused less into adjacent muscle tissue than BTX, indicating that INI101 toxin may reduce potential side effects due to diffusion into normal tissues. INI101 toxin isolated from the novel strain CCUG 7968 is a newly identified meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field. KEY POINTS: • Botulinum neurotoxin type A (BoNT/A, INI101) was identified from the CCUG 7968 strain. • INI101 toxin showed similar safety and therapeutic efficacy comparable to conventional BoNT/A both in vitro and in vivo. • INI101 toxin is a meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field.

Anticancer effects of oleanolic acid on human melanoma cells.

Owing to the ineffectiveness of the currently used therapies against melanoma, there has been a shift in focus toward alternative therapies involving the use of natural compounds. This study assessed the anticancer effects of oleanolic acid (OA) and its ability to induce apoptosis in A375SM and A375P melanoma cells in vivo. Compared to the control group, viability of A375P and A375SM cells decreased following OA treatment. In OA-treated A375SM and A375P cells, 4',6-diamidino-2-phenylindole staining showed an increase in the apoptotic body, and flow cytometry revealed increased number of apoptotic cells compared to that in the control group. OA-treated A375SM cells exhibited an increased expression of the apoptotic proteins, cleaved poly (ADP-ribose) polymerase (PARP) and B-cell lymphoma (Bcl)-2-associated X protein (Bax) as well as decreased expression of the antiapoptotic protein Bcl-2 compared to that in the control group. In OA-treated A375P cells, expression patterns of cleaved PARP and Bcl-2 were similar to those in OA-treated A375SM cells; however, no difference was reported in the expression of Bax compared to that in the control group. Additionally, OA-treated melanoma cells showed decreased expression of phospho-nuclear factor-κB (p-NF-κB), phospho-inhibitor of nuclear factor-κBα (p-IκBα), and phospho-IκB kinase αß than that in the control group. Moreover, immunohistochemistry showed a comparatively decreased level of p-NF-κB in the OA-treated group than that in the control group. Xenograft analysis confirmed the in vivo anticancer effects of OA against A375SM cells. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay revealed an increased number of TUNEL-positive cells in the OA-treated group compared to that in the control group. In conclusion, the study results suggest that OA induces apoptosis of A375SM and A375P cells in vitro and apoptosis of A375SM cells in vivo. Furthermore, the in vitro and in vivo anticancer effects were mediated by the NF-κB pathway.

Silymarin inhibits proliferation of human breast cancer cells via regulation of the MAPK signaling pathway and induction of apoptosis.

Silymarin is a purified mixture of four isomeric flavonoids extracted from the seeds and fruit of the milk thistle plant, Silybum marianus (L.). Silymarin exhibits a wide variety of biological effects and is commonly used in traditional medicine. Therefore, the anticancer effects of silymarin on human breast cancer cells were investigated to determine its pharmacological mechanisms in vitro and in vivo. The viability and proliferation of MDA-MB- 231 and MCF-7 breast cancer cells were investigated using MTT and wound healing assays. Silymarin decreased the viability and proliferation of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner. The number of apoptotic bodies, as shown by DAPI staining, was increased in a concentration-dependent manner, indicating that silymarin induces apoptosis. Additionally, changes in the expression levels of apoptosis-related proteins were demonstrated in human breast cancer cells using western blotting. Silymarin increased the levels of Bax, cleaved poly-ADP ribose polymerase, cleaved caspase-9 and phosphorylated (p-)JNK, and decreased the levels of Bcl-2, p-P38 and p-ERK1/2. Furthermore, the inhibitory effects of silymarin on MCF-7 tumor growth were investigated. In mice treated with silymarin for 3 weeks (25 and 50 mg/kg), MCF-7 tumor growth was inhibited without organ toxicity. In MCF-7 tumors, silymarin induced apoptosis and decreased p-ERK1/2 levels, as assessed using a TUNEL assay and immunohistochemistry. These results indicated that silymarin inhibited breast cancer cell proliferation both in vitro and in vivo by modulating the MAPK signaling pathway. Therefore, silymarin may potentially be used as a chemo-preventive or therapeutic agent.

The atypical antipsychotic risperidone targets hypothalamic melanocortin 4 receptors to cause weight gain.

Atypical antipsychotics such as risperidone cause drug-induced metabolic syndrome. However, the underlying mechanisms remain largely unknown. Here, we report a new mouse model that reliably reproduces risperidone-induced weight gain, adiposity, and glucose intolerance. We found that risperidone treatment acutely altered energy balance in C57BL/6 mice and that hyperphagia accounted for most of the weight gain. Transcriptomic analyses in the hypothalamus of risperidone-fed mice revealed that risperidone treatment reduced the expression of Mc4r. Furthermore, Mc4r in Sim1 neurons was necessary for risperidone-induced hyperphagia and weight gain. Moreover, we found that the same pathway underlies the obesogenic effect of olanzapine-another commonly prescribed antipsychotic drug. Remarkably, whole-cell patch-clamp recording demonstrated that risperidone acutely inhibited the activity of hypothalamic Mc4r neurons via the opening of a postsynaptic potassium conductance. Finally, we showed that treatment with setmelanotide, an MC4R-specific agonist, mitigated hyperphagia and obesity in both risperidone- and olanzapine-fed mice.

Neuroendocrine control of appetite and metabolism.

Body homeostasis is predominantly controlled by hormones secreted by endocrine organs. The central nervous system contains several important endocrine structures, including the hypothalamic-pituitary axis. Conventionally, neurohormones released by the hypothalamus and the pituitary gland (hypophysis) have received much attention owing to the unique functions of the end hormones released by their target peripheral organs (e.g., glucocorticoids released by the adrenal glands). Recent advances in mouse genetics have revealed several important metabolic functions of hypothalamic neurohormone-expressing cells, many of which are not readily explained by the action of the corresponding classical downstream hormones. Notably, the newly identified functions are better explained by the action of conventional neurotransmitters (e.g., glutamate and GABA) that constitute a neuronal circuit. In this review, we discuss the regulation of appetite and metabolism by hypothalamic neurohormone-expressing cells, with a focus on the distinct contributions of neurohormones and neurotransmitters released by these neurons.

Apigenin induces apoptosis by regulating Akt and MAPK pathways in human melanoma cell A375SM.

Apigenin, an aromatic compound, exhibits antioxidant, anti­inflammatory and anti­viral effects. The present study aimed to investigate the effects of apigenin on cell proliferation and apoptosis of human melanoma cells A375P and A375SM. Therefore, melanoma cells were treated with apigenin to determine its anti­proliferative and survival effects, using wound healing and MTT assays. The results revealed that melanoma cell viability was decreased in a dose­dependent manner. Furthermore, chromatin condensation, indicating apoptosis, was significantly increased in a dose­dependent manner, as demonstrated by DAPI staining. In addition, increased apoptosis rate following treatment with apigenin was confirmed by Annexin V­propidium iodide staining. The changes in the expression levels of apoptosis­related proteins in A375P and A375SM melanoma cells were subsequently detected using western blot analysis. The results demonstrated that the protein expression levels of Bcl­2 were decreased, whereas those of Bax, cleaved poly ADP­ribose polymerase, cleaved caspase­9 and p53 were upregulated in a dose­dependent manner in apigenin­treated cells compared with those noted in untreated cells. In addition, in apigenin­treated A375P cells, phosphorylated (p)­p38 was upregulated and p­extracellular signal­regulated kinase (ERK), p­c­Jun N­terminal kinase (JNK) and p­protein kinase B (Akt) were downregulated. However, in A375SM cells, apigenin treatment increased p­ERK and p­JNK and decreased p­p38 and p­Akt protein expression levels. Subsequently, the inhibitory effect of apigenin on tumor growth was investigated in vivo. Tumor volume was significantly reduced in the 25 and 50 mg/kg apigenin­treated groups compared with the control group. Additionally, a TUNEL assay was performed to detect apoptotic cells. Immunohistochemical staining also revealed elevated p­ERK expression in the apigenin­treated group compared with the control group. Overall, the findings of the present study indicated that apigenin attenuated the growth of A375SM melanoma cells by inducing apoptosis via regulating the Akt and mitogen­activated protein kinase signaling pathways.

Targeting X chromosome-linked inhibitor of apoptosis protein in mucoepidermoid carcinoma of the head and neck: A novel therapeutic strategy using nitidine chloride.

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

Silymarin induces inhibition of growth and apoptosis through modulation of the MAPK signaling pathway in AGS human gastric cancer cells.

Apoptosis is regarded as a therapeutic target because it is typically disturbed in human cancer. Silymarin from milk thistle (Silybum marianum) has been reported to exhibit anticancer properties via regulation of apoptosis as well as anti­inflammatory, antioxidant and hepatoprotective effects. In the present study, the effects of silymarin on the inhibition of proliferation and apoptosis were examined in human gastric cancer cells. The viability of AGS human gastric cancer cells was assessed by MTT assay. The migration of AGS cells was investigated by wound healing assay. Silymarin was revealed to significantly decrease viability and migration of AGS cells in a concentration­dependent manner. In addition, the number of apoptotic bodies and the rate of apoptosis were increased in a dose­dependent manner as determined by DAPI staining and Annexin V/propidium iodide double staining. The changes in the expression of silymarin­induced apoptosis proteins were investigated in human gastric cancer cells by western blotting analysis. Silymarin increased the expression of Bax, phosphorylated (p)­JNK and p­p38, and cleaved poly­ADP ribose polymerase, and decreased the levels of Bcl­2 and p­ERK1/2 in a concentration­dependent manner. The in vivo tumor growth inhibitory effect of silymarin was investigated. Silymarin (100 mg/kg) significantly decreased the AGS tumor volume and increased apoptosis, as assessed by the TUNEL assay, confirming its tumor­inhibitory effect. Immunohistochemical staining revealed elevated expression of p­JNK and p­p38 as well as reduced expression of p­ERK1/2 associated with silymarin­treatment. Silymarin was revealed to reduce tumor growth through inhibition of p­ERK and activation of p­p38 and p­JNK in human gastric cancer cells. These results indicated that silymarin has potential for development as a cancer therapeutic due to its growth inhibitory effects and induction of apoptosis in human gastric cancer cells.

Antitumor and apoptotic effects of quercetin on human melanoma cells involving JNK/P38 MAPK signaling activation.

In this study, we investigated whether Quercetin has anti-cancer effects on A375SM and A375P human melanoma cells. Cell viability was assessed using an MTT assay. The proliferation of melanoma cells was measured by a wound-healing assay. Quercetin significantly decreased viability and proliferation of A375SM cells in a concentration-dependent manner. However, quercetin had no effect on A375P cells. DAPI staining showed increased chromatin condensation in a concentration-dependent manner, indicating apoptosis. Flow cytometric analysis indicated that quercetin suppressed the viability of A375SM cells by inducing apoptosis. Expression of quercetin-induced apoptosis proteins was investigated by Western blot analysis. Quercetin increased the expression of Bax, phospho-JNK, phospho-p38 and phospho-ERK1/2, cleaved poly-ADP ribose polymerase and decreased Bcl-2 in a concentration-dependent manner. We also investigated the in vivo tumor-growth inhibitory effect of quercetin. Quercetin (at 50 and 100 mg/kg) significantly decreased the A375SM tumor volume compared to the control group and increased apoptosis as assessed by the TUNEL assay. Immunohistochemistry staining revealed that the level of phosphor-JNK and phosphor-p38 increased in the quercetin-treated mice. These results indicate that quercetin inhibited the growth of A375SM melanoma cells through apoptosis and thus can be regarded as a new and effective chemo-preventive or therapeutic agent.

ABT-263 exhibits apoptosis-inducing potential in oral cancer cells by targeting C/EBP-homologous protein.

PURPOSE: ABT-263 is a potent BH3 mimetic that possesses anticancer potential against various types of cancer. In general, this potential is due to its high binding affinity to anti-apoptotic proteins in the Bcl-2 family that disrupt sequestration of pro-apoptotic proteins. In the present study, we sought to identify an alternative regulatory mechanism responsible for ABT-263-mediated anticancer activity in human oral cancer. METHODS: We investigated the in vitro anti-cancer effects of ABT-263 using a trypan blue exclusion assay, Western blotting, DAPI staining, immunofluorescence staining, a live/dead assay, microarray-based expression profiling, and quantitative real-time PCR. In vivo anti-tumorigenic effects of ABT-263 were examined using a nude mouse tumor xenograft model, a TUNEL assay, and immunohistochemistry. RESULTS: We found that ABT-263 suppressed viability and induced apoptosis in human oral cancer-derived cell lines HSC-3 and HSC-4. Subsequent microarray-based gene expression profiling revealed 55 differentially expressed genes in the ABT-263-treatead group, including 12 genes associated with "endoplasmic reticulum stress and apoptosis." Consistent with the microarray results, the mRNA expression levels of the top four genes (CHOP, TRB3, ASNS, and STC2) were found to be significantly increased. In addition, we found that ABT-263 considerably enhanced the expression levels of the C/EBP-homologous protein (CHOP) and its mRNA, resulting in apoptosis induction in four other human oral cancer-derived cell lines (MC-3, YD-15, HN22, and Ca9.22). Extending our in vitro findings, we found that ABT-263 reduced the growth of HSC-4 cells in vivo at a dosage of 100 mg/kg/day without any change in body weight. TUNEL-positive cells were also found to be increased in tumors of ABT-263-treated mice without any apparent histopathological changes in liver or kidney tissues. CONCLUSIONS: These results provide evidence that ABT-263 may serve as an effective therapeutic agent for the treatment of human oral cancer.