Metallothionein Family Proteins as Regulators of Zinc Ions Synergistically Enhance the Anticancer Effect of Cannabidiol in Human Colorectal Cancer Cells.

Cannabidiol (CBD) is a chemical obtained from Cannabis sativa; it has therapeutic effects on anxiety and cognition and anti-inflammatory properties. Although pharmacological applications of CBD in many types of tumors have recently been reported, the mechanism of action of CBD is not yet fully understood. In this study, we perform an mRNA-seq analysis to identify the target genes of CBD after determining the cytotoxic concentrations of CBD using an MTT assay. CBD treatment regulated the expression of genes related to DNA repair and cell division, with metallothionein (MT) family genes being identified as having highly increased expression levels induced by CBD. It was also found that the expression levels of MT family genes were decreased in colorectal cancer tissues compared to those in normal tissues, indicating that the downregulation of MT family genes might be highly associated with colorectal tumor progression. A qPCR experiment revealed that the expression levels of MT family genes were increased by CBD. Moreover, MT family genes were regulated by CBD or crude extract but not by other cannabinoids, suggesting that the expression of MT family genes was specifically induced by CBD. A synergistic effect between CBD and MT gene transfection or zinc ion treatment was found. In conclusion, MT family genes as novel target genes could synergistically increase the anticancer activity of CBD by regulating the zinc ions in human colorectal cancer cells.

Knockout of FosB gene changes drug sensitivity and invasion activity via the regulation of Bcl-2, E-cadherin, ß-catenin, and vimentin expression.

Our previous research suggested the presence of a novel SETDB1-mediated FosB pathway that could be responsible for the regulation of cell proliferation and invasiveness during anticancer treatments. In this study, we prepared FosB knock-out (FosB-KO) A549 human lung cancer cells using the CRISPR/Cas9 system and examined the physiological and molecular changes that caused. Annexin V and TUNEL assays showed that FosB-KO clones were less sensitive to doxorubicin treatment compared to the control A549 cells. Bcl2 expression and mitochondrial membrane potential were also both markedly increased in FosB-KO clones, which suggests the involvement of Bcl2 in the doxorubicin mediated increase in cell viability demonstrated the FosB-KO clones. Moreover, we identified changes in the migration and transforming activities of the FosB-KO clones that coincided with changes in the expression levels of E-cadherin, ß-catenin, and Vimentin. RT-PCR and qPCR analysis showed that the expressions of Bcl2, E-cadherin, ß-catenin, and Vimentin were regulated at the transcriptional level. Importantly, FosB overexpression in FosB-KO clones restored the expression of Bcl2, Akt, E-cad, ß-catenin, and Vimentin, suggesting that those proteins were tightly regulated by FosB. These data suggest that the FosB gene critically regulates both drug sensitivity and invasion related genes, and does so in a manner coordinated with the function of SETDB1. Therefore, we propose that the FosB gene regulates both drug sensitivity and invasion activity related genes, and also shows coordinated function with SETDB1 for the regulation of target proteins.

Knockout of SETDB1 gene using the CRISPR/cas-9 system increases migration and transforming activities via complex regulations of E-cadherin, ß-catenin, STAT3, and Akt.

SETDB1 HMTase participates in various cellular processes via epigenetic transcriptional regulation. SETDB1 expression is downregulated by anticancer drug treatment in cancer cells, but we still need to verify the functional significance on SETDB1 downregulation. CRISPR/cas9 is a useful technology for doing a knockout (KO) of a target gene. It is widely used to examine the function of genes. In this study, we prepared SETDB1-KO from A549 human lung cancer cells using the CRISPR/Cas9 system, and we compared molecular changes between the A549 cells and the SETDB1-KO cells. The SETDB1-KO cell proliferation rate was slightly decreased as compared to the A549 cells, but there was no large difference in sensitivity with doxorubicin treatment. Instead, the migration activity and transforming activity were dramatically increased in SETDB-KO cells. Using a western blot analysis and an immunostaining experiment, we confirmed that SETDB1-KO downregulates the expression of E-cadherin and ß-catenin. A qPCR and an RT-PCR analysis suggested that SETDB1 transcriptionally regulates E-cadherin and ß-catenin. Moreover, E-cadherin expression was also detected in the cytoplasmic region of SETDB1-KO cells, indicating that functional localization of E-cadherin might be changed in SETDB1-KO cells. On the other hand, total levels of STAT3 and Akt were increased in the SETDB1-KO cells, but activation of STAT3 (pSTAT3) was not induced in doxorubicin-treated SETDB1-KO cells. SETDB1 overexpression into SETDB1-KO cells restores the expression of E-cadherin, ß-catenin, STAT3, and Akt, suggesting that those proteins are tightly regulated by SETDB1. Collectively, we suggest that complex regulations on E-cadherin, ß-catenin, STAT3, and Akt are correlated with the increased migration and transforming activity of SETDB1-KO cells.

SETDB1-mediated FosB regulation via ERK2 is associated with an increase in cell invasiveness during anticancer drug treatment of A549 human lung cancer cells.

We have determined a functional link to the inverse expression of SETDB1 and FosB following anticancer drug treatment. Doxorubicin treatment caused decreased SETDB1 expression and FosB overexpression both at the mRNA and protein levels. The decreased HMTase activity of SETDB1 coincided with altered occupancy across the promoter region of the FosB gene. SETDB1 overexpression decreased the luciferase reporter activity containing the FosB promoter region, but siSETDB1 increased the luciferase reporter activity, suggesting that SETDB1 directly and negatively regulated FosB expression. In addition, MEK inhibitor (PD98059) blocked the SETDB1 regulation of the FosB promoter activity via ERK2 activation during doxorubicin treatment. A microscopic analysis reveals that FosB expression was observed in living cells in spite of doxorubicin treatment. Ectopic FosB/ΔFosB expression increased the number of colonies and the migration of A549 cells compared to that in control. These results suggest that the ERK2-SETDB1-FosB signaling pathway might have an anti-therapeutic regulatory mechanism that increases the transformation and migration activity of cancer cells during anticancer drug treatment.

Cell death induction and intracellular vesicle formation in human colorectal cancer cells treated with Δ9-Tetrahydrocannabinol.

BACKGROUND: Δ9-Tetrahydrocannabinol (Δ9-THC) is a principal psychoactive extract of Cannabis sativa and has been traditionally used as palliative medicine for neuropathic pain. Cannabidiol (CBD), an extract of hemp species, has recently attracted increased attention as a cancer treatment, but Δ9-THC is also requiring explored pharmacological application. OBJECTIVE: This study evaluated the pharmacological effects of Δ9-THC in two human colorectal cancer cell lines. We investigated whether Δ9-THC treatment induces cell death in human colorectal cancer cells. METHODS: We performed an MTT assay to determine the pharmacological concentration of Δ9-THC. Annxein V and Western blot analysis confirmed that Δ9-THC induced apoptosis in colorectal cancer cells. Metabolic activity was evaluated using MitoTracker staining and ATP determination. We investigated vesicle formation by Δ9-THC treatment using GW9662, known as a PPARγ inhibitor. RESULTS: The MTT assay showed that treatment with 40 µM Δ9-THC and above inhibited the proliferation of colorectal cancer cells. Multiple intracytoplasmic vesicles were detected upon microscopic observation, and fluorescence-activated cell sorting analysis showed cell death via G1 arrest. Δ9-THC treatment increased the expression of cell death marker proteins, including p53, cleaved PARP-1, RIP1, and RIP3, suggesting that Δ9-THC induced the death of colorectal cancer cells. Δ9-THC treatment also reduced ATP production via changes in Bax and Bcl-2. Δ9-THC regulated intracytoplasmic vesicle formation by modulating the expression of PPARγ and clathrin, adding that antiproliferative activity of Δ9-THC was also affected. CONCLUSION: In conclusion, Δ9-THC regulated two functional mechanisms, intracellular vesicle formation and cell death. These findings can help to determine how cannabinoids can be used most effectively to improve the efficacy of cancer treatment.

Dual Cytotoxic Responses Induced by Treatment of A549 Human Lung Cancer Cells with Sweet Bee Venom in a Dose-Dependent Manner.

Objectives: Sweet bee venom (sBV) is purified from Apis mellifera, containing a high level of melittin-its main component. It has been used as a therapeutic agent for pain relief and anti-inflammation, as well as for treating neuronal abnormalities. Recently, there have been studies on the therapeutic application of sBV for anticancer treatment. In the present study, we investigated the pharmacological effect of sBV treatment in A549 human lung cancer cells. Methods: We used microscopic analysis to observe the morphological changes in A549 cells after sBV treatment. The MTT assay was used to examine the cytotoxic effect after dose-dependent sBV treatment. Molecular changes in sBV were evaluated by the expression of apoptosis marker proteins using western blot analysis. Results: Microscopic analysis suggested that the growth inhibitory effect occurred in a dose-dependent manner; however, cell lysis occurred at a concentration over 20 µg/mL of sBV. The MTT assay indicated that sBV treatment exhibited a growth inhibitory effect at a concentration over 5 µg/mL. On fluorescence activated cell sorting analysis, G0 dead cells were observed after G1 arrest at treatment concentrations up to 10 µg/mL. However, rapid cell rupture was observed at a concentration of 20 µg/mL. Western blot analysis demonstrated that sBV treatment modulated the expression of multiple cell death-related proteins, including cleaved-PARP, cleaved-caspase 9, p53, Bcl2, and Bax. Conclusion: sBV induced cell death in A549 human lung cancer cells at a pharmacological concentration, albeit causing hemolytic cell death at a high concentration.

Sweet Bee Venom Triggers Multiple Cell Death Pathways or Spurs Acute Cell Rupture According to Its Concentration in THP-1 Monocytic Leukemia Cells.

Sweet bee venom (sBV) contains various pharmacologically active components of bee venom (BV), but it is modified via the removal of the harmful substances found in BV. Thus, sBV has been used for pain relief in Oriental medicine but has only recently been applied for the treatment of various diseases. In this study, we examined the pharmacological effects and immunomodulatory functions of sBV in THP-1 monocytic leukemia cells. Growth inhibition and cell death were observed according to the concentration of sBV. However, the rapid collapse of cell cycle distribution was shown at 20 µg/mL sBV treatment, indicating that sBV led to cell death or acute cell rupture according to concentration. sBV administration activated Caspase-9, PARP1, RIPK1, and RIPK3, suggesting that the pharmacological actions of sBV were associated with induction of apoptosis and necroptosis. On the other hand, sBV or LPS administration increased cytokine expression, including IL-1ß, and showed synergistic cell death in combinatory treatment conditions. Moreover, combinatory administration of sBV and LPS induced severe damage or death during egg development. This result implies that sBV exhibits both pharmacological and toxic effects depending on its concentration. Therefore, sBV might be a promising therapeutic approach, but optimal concentration should be considered before treatment.

Cannabidiol Regulates PPARγ-Dependent Vesicle Formation as well as Cell Death in A549 Human Lung Cancer Cells.

Extracts of phytocannabinoids from Cannabis sativa have been studied for therapeutic purposes. Although nonpsychoactive CBD has been studied as a promising anticancer drug because it induces apoptosis in many cancer cells, it is also known to induce several physiological changes. In this study, we clarify the functional role it plays in the morphological characteristics of intracellular vesicle formation as well as apoptosis in A549 human lung cancer cells. CBD treatment shows growth inhibition at concentrations above 20 µM, but FACS analysis shows low efficacy in terms of cell death. Microscopic observations suggest that multiple vesicles were detected in the cytoplasmic region of CBD-treated A549 cells. CBD treatment upregulates apoptosis-related proteins, such as p53, PARP, RIP1, RIP3, Atg12, and Beclin, indicating that CBD regulates several types of cell death. CBD treatment also induced E-cadherin, PPARγ, clathrin, ß-adaptin, and Tsg101, also known to be cellular-differentiation inducers or vesicle-formation components. Treatment combining CBD with GW9662, a PPARγ inhibitor, reduced CBD-induced cytoplasmic vesicle formation. This indicates that PPARγ regulates the vesicle-formation mechanism. However, CBD-treated E-cad KO clones did not show this regulatory mechanism. These results elucidate the pharmacological and molecular networks associated with CBD in PPARγ-dependent vesicle formation and the induction of apoptosis.

Identification of Cleaved Haptoglobin in the Serum of Bee Venom-Hypersensitive Patients.

Background: Bee venom has been used as a therapeutic compound for various human diseases in oriental medicine; however, it can induce anaphylaxis in hypersensitive patients during treatment. Anaphylaxis is an acute allergic reaction that occurs after allergen exposure. IgE is released from immune-related cells such as mast cells and basophils during anaphylaxis. Various inflammatory mediators are also released into the bloodstream during the acute response. Objectives: We aimed to identify specific proteins from bee venom-hypersensitive patients. Methods: We analyzed the blood serum of control and bee venom-hypersensitive patients using two-dimensional (2D) electrophoresis. Results: An interesting protein spot with a molecular size of 10 kDa was identified at an isoelectric point (p.I.) of 5.5. Spots detected both before and after sweet bee venom therapy were not proteins induced by sweet bee venom. The 10 kDa protein was identified as the cleaved form of haptoglobin through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Statistical analysis indicated that the presence of the spot was highly significant in the bee venom-hypersensitive group. Conclusion: The findings suggest that cleaved haptoglobin may be a significant diagnostic protein for anaphylaxis. In addition, a high incidence of bee venom hypersensitivity may be associated with the haptoglobin genotype.

Regulatory Effect of Cannabidiol (CBD) on Decreased ß-Catenin Expression in Alopecia Models by Testosterone and PMA Treatment in Dermal Papilla Cells.

OBJECTIVES: The hair follicle is composed of more than 20 kinds of cells, and mesoderm derived dermal papilla cells and keratinocytes cooperatively contribute hair growth via Wnt/ß-catenin signaling pathway. We are to investigate ß-catenin expression and regulatory mechanism by CBD in alopecia hair tissues and dermal papilla cells. METHODS: We performed structural and anatomical analyses on alopecia patients derived hair tissues using microscopes. Pharmacological effect of CBD was evaluated by ß-catenin expression using RT-PCR and immunostaining experiment. RESULTS: Morphological deformation and loss of cell numbers in hair shaft were observed in alopecia hair tissues. IHC experiment showed that loss of ß-catenin expression was shown in inner shaft of the alopecia hair tissues, indicating that ß-catenin expression is a key regulatory function during alopecia progression. Consistently, ß-catenin expression was decreased in testosterone or PMA treated dermal papilla cells, suggesting that those treatments are referred as a model on molecular mechanism of alopecia using dermal papilla cells. RT-PCR and immunostaining experiments showed that ß-catenin expression was decreased in RNA level, as well as decreased ß-catenin protein might be resulted from ubiquitination. However, CBD treatment has no changes in gene expression including ß-catenin, but the decreased ß-catenin expression by testosterone or PMA was restored by CBD pretreatment, suggesting that potential regulatory effect on alopecia induction of testosterone and PMA. CONCLUSION: CBD might have a modulating function on alopecia caused by hormonal or excess of signaling pathway, and be a promising application for on alopecia treatment.

Increased Expression of FosB through Reactive Oxygen Species Accumulation Functions as Pro-Apoptotic Protein in Piperlongumine Treated MCF7 Breast Cancer Cells.

Piperlongumine (PL), a natural alkaloid compound isolated from long pepper (Piper longum), can selectively kill cancer cells, but not normal cells, by accumulation of reactive oxygen species (ROS). The objective of this study was to investigate functional roles of expression of SETDB1 and FosB during PL treatment in MCF7 breast cancer cells. PL downregulates SETDB1 expression, and decreased SETDB1 expression enhanced caspase 9 dependent-PARP cleavage during PL-induced cell death. PL treatment generated ROS. ROS inhibitor NAC (N-acetyl cysteine) recovered SETDB1 expression decreased by PL. Decreased SETDB1 expression induced transcriptional activity of FosB during PL treatment. PARP cleavage and positive annexin V level were increased during PL treatment with FosB overexpression whereas PARP cleavage and positive annexin V level were decreased during PL treatment with siFosB transfection, implying that FosB might be a pro-apoptotic protein for induction of cell death in PL-treated MCF7 breast cancer cells. PL induced cell death in A549 lung cancer cells, but molecular changes involved in the induction of these cell deaths might be different. These results suggest that SETDB1 mediated FosB expression may induce cell death in PL-treated MCF7 breast cancer cells.

Homeostatic balance of histone acetylation and deconstruction of repressive chromatin marker H3K9me3 during adipocyte differentiation of 3T3-L1 cells.

Background Adipocyte differentiation is completed by changing gene expression. Chromatin is closely related to gene expression. Therefore, its structure might be changed for adipocyte differentiation. Mouse 3T3-L1 preadipocytes have been used as a cell model to study molecular mechanisms of adipogenesis. Objective To examine changes of chromatin modification and expression of histone modifying enzymes during adipocyte differentiation. Methods Microscopic analysis and Oil Red O staining were performed to determine distinct phenotype of adipocyte differentiation. RT-PCR and Western blot analysis were used to examine expression levels of histone modifying enzymes during adipocyte differentiation. Histone modifications were examined by immunostaining analysis. Results Expression levels of P300 and cbp were increased during adipocyte differentiation. However, acetylation of histones was not quantitatively changed postdifferentiation of 3T3-L1 cells compared to that at pre-differentiation. RT-PCR and Western blot analyses showed that expression levels of hdac2 and hdac3 were increased during adipocyte differentiation, suggesting histone acetylation at chromatin level was homeostatically controlled by increased expression of both HATs and HDACs. Tri-methylation level of H3K9 (H3K9me3), but not that of H3K27me3, was significantly decreased during adipocyte differentiation. Decreased expression of setdb1 was consistent with reduced pattern of H3K9me3. Knock-down of setdb1 induced adipocyte differentiation. This suggests that setdb1 is a key chromatin modifier that modulates repressive chromatin. Conclusion These results suggest that there exist extensive mechanisms of chromatin modifications for homeostatic balance of chromatin acetylation and deconstruction of repressive chromatin during adipocyte differentiation.

SETDB1 mediated FosB expression increases the cell proliferation rate during anticancer drug therapy.

The efficacy of anticancer drugs depends on a variety of signaling pathways, which can be positively or negatively regulated. In this study, we show that SETDB1 HMTase is down-regulated at the transcriptional level by several anticancer drugs, due to its inherent instability. Using RNA sequence analysis, we identified FosB as being regulated by SETDB1 during anticancer drug therapy. FosB expression was increased by treatment with doxorubicin, taxol and siSETDB1. Moreover, FosB was associated with an increased rate of proliferation. Combinatory transfection of siFosB and siSETDB1 was slightly increased compared to transfection of siFosB. Furthermore, FosB was regulated by multiple kinase pathways. ChIP analysis showed that SETDB1 and H3K9me3 interact with a specific region of the FosB promoter. These results suggest that SETDB1- mediated FosB expression is a common molecular phenomenon, and might be a novel pathway responsible for the increase in cell proliferation that frequently occurs during anticancer drug therapy. [BMB Reports 2016; 49(4): 238-243].