Hironori Aramaki

Cannabidiolic acid, a major cannabinoid in fiber-type cannabis, is an inhibitor of MDA-MB-231 breast cancer cell migration

Cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, has been reported to possess diverse biological activities, including anti-proliferative effect on cancer cells. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), few studies have investigated whether CBDA itself is biologically active. Results of the current investigation revealed that CBDA inhibits migration of the highly invasive MDA-MB-231 human breast cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. It is established that activation of the RhoA signaling pathway leads to inhibition of the mobility of various cancer cells, including MDA-MB-231 cells. The data presented in this report suggest for the first time that as an active component in the cannabis plant, CBDA offers potential therapeutic modality in the abrogation of cancer cell migration, including aggressive breast cancers.

(-)-Xanthatin selectively induces GADD45γ and stimulates caspase-independent cell death in human breast cancer MDA-MB-231 cells

exo-Methylene lactone group-containing compounds, such as (--)-xanthatin, are present in a large variety of biologically active natural products, including extracts of Xanthium strumarium (Cocklebur). These substances are reported to possess diverse functional activities, exhibiting anti-inflammatory, antimalarial, and anticancer potential. In this study, we synthesized six structurally related xanthanolides containing exo-methylene lactone moieties, including (--)-xanthatin and (+)-8-epi-xanthatin, and examined the effects of these chemically defined substances on the highly aggressive and farnesyltransferase inhibitor (FTI)-resistant MDA-MB-231 cancer cell line. The results obtained demonstrate that (--)-xanthatin was a highly effective inhibitor of MDA-MB-231 cell growth, inducing caspase-independent cell death, and that these effects were independent of FTase inhibition. Further, our results show that among the GADD45 isoforms, GADD45γ was selectively induced by (--)-xanthatin and that GADD45γ-primed JNK and p38 signaling pathways are, at least in part, involved in mediating the growth inhibition and potential anticancer activities of this agent. Given that GADD45γ is becoming increasingly recognized for its tumor suppressor function, the results presented here suggest the novel possibility that (--)-xanthatin may have therapeutic value as a selective inducer of GADD45γ in human cancer cells, in particular in FTI-resistant aggressive breast cancers.

Δ9-Tetrahydrocannabinol Disrupts Estrogen-Signaling through Up-Regulation of Estrogen Receptor β (ERβ)

Δ(9)-Tetrahydrocannabinol (Δ(9)-THC) has been reported as possessing antiestrogenic activity, although the mechanisms underlying these effects are poorly delineated. In this study, we used the estrogen receptor α (ERα)-positive human breast cancer cell line, MCF-7, as an experimental model and showed that Δ(9)-THC exposures markedly suppresses 17β-estradiol (E2)- induced MCF-7 cell proliferation. We demonstrate that these effects result from Δ(9)-THCs ability to inhibit E2-liganded ERα activation. Mechanistically, the data obtained from biochemical analyses revealed that (i) Δ(9)-THC up-regulates ERβ, a repressor of ERα, inhibiting the expression of E2/ERα-regulated genes that promote cell growth and that (ii) Δ(9)-THC induction of ERβ modulates E2/ERα signaling in the absence of direct interaction with the E2 ligand binding site. Therefore, the data presented support the concept that Δ(9)-THCs antiestrogenic activities are mediated by the ERβ disruption of E2/ERα signaling.

Effect of Dosing Schedule on Pharmacokinetics of Alpha Interferon and Anti-Alpha Interferon Neutralizing Antibody in Mice

ABSTRACT The influences of dosing time and dosing schedule on the plasma alpha interferon (IFN-α) concentration and the production of anti-IFN-α neutralizing antibodies were investigated in ICR male mice adapted to cycles of 12 h of light and 12 h of dark. In mice pretreated with IFN-α for 21 days, the plasma IFN-α concentrations were significantly lower than those in control mice (P< 0.01). The clearance of IFN-α and its volume of distribution obtained at steady state were significantly higher in the animals with IFN-α pretreatment than in the mice without IFN-α pretreatment. The area under the concentration-time curve and the mean residence time of IFN-α were significantly smaller in IFN-α-pretreated animals than in control animals. The plasma IFN-α levels (measured 2 h after dosing) were significantly lower in mice treated daily with IFN-α, while the anti-IFN-α neutralizing antibody levels (measured 24 h after dosing) were significantly increased on days 15 and 21 of treatment. Plasma IFN-α levels were significantly decreased in association with the production of anti-IFN-α neutralizing antibodies in mice treated with IFN-α daily at either 0900 or 2100 h. By contrast, the plasma IFN-α levels (measured 2 h after dosing) remained stable in mice treated with IFN-α at 0900 h on alternate days, while they were significantly lower after 21 days of treatment in mice treated with IFN-α at 2100 h on alternate days. These changes were associated with a significant increase in the levels of anti-IFN-α neutralizing antibodies in the latter group. The present findings suggest that an appropriate dosing schedule and/or dosing time for IFN-α may reduce the level of production of anti-IFN-α neutralizing antibodies in experimental and clinical situations.

(−)-Xanthatin up-regulation of the GADD45γ tumor suppressor gene in MDA-MB-231 breast cancer cells: Role of topoisomerase IIα inhibition and reactive oxygen species

Previously, we reported that (-)-xanthatin, a naturally occurring xanthanolide present in the Cocklebur plant, exhibits potent anti-proliferative effects on human breast cancer cells, accompanied by an induction of the growth arrest and DNA damage-inducible gene 45γ (GADD45γ), recognized recently as a novel tumor suppressor gene. However, the mechanisms mediating this activation were unknown. Topoisomerase IIα (Topo IIα) inhibition has been reported to produce a cell death response accompanied by an atypical DNA laddering fragmentation profile, similar to that noted previously for (-)-xanthatin. Therefore we hypothesized that (-)-xanthatins GADD45γ activation was mediated through the Topo IIα pathway. Here, we identify that (-)-xanthatin does function as a catalytic inhibitor of Topo IIα, promoting DNA damage. In addition, reactive oxygen species (ROS) were elevated in cells treated with this agent. Mechanistically, it was determined that the induced levels of GADD45γ mRNA resulting from (-)-xanthatin exposures were stabilized by coordinately produced ROS, and that the consequent induction of GADD45γ mRNA, GADD45γ protein and ROS generation were abrogated by co-treatment with N-acetyl-l-cysteine. Taken together, the data support the concept that Topo IIα inhibition by (-)-xanthatin is a trigger that stimulates expression of DNA damage-inducible GADD45γ mRNA and that concomitantly produced ROS act downstream to further enhance the GADD45γ mRNA/GADD45γ protein induction process, resulting in breast cancer cell death.

Δ9-THC modulation of fatty acid 2-hydroxylase (FA2H) gene expression: Possible involvement of induced levels of PPARα in MDA-MB-231 breast cancer cells

We recently reported that Δ(9)-tetrahydrocannabinol (Δ(9)-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2-hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ(9)-THC-treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ(9)-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA-MB-231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ(9)-THC-mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ(9)-THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ(9)-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ(9)-THC-induced up-regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ(9)-THC up-regulation of FA2H in MDA-MB-231 cells.

Dosing schedule-dependent change in the disruptive effects of interferon-α on the circadian clock function

Altered homeostatic regulation, including the disturbance of circadian rhythms, is often observed in patients undergoing interferon (IFN) therapy. We reported previously that IFN-alpha has the ability to modulate the circadian clock function at the molecular level and that the alteration of clock function could be overcome by changing the dosing schedule. In this study, we investigated the influence of IFN-alpha on the intrinsic biological rhythms in mice by comparing two dosing schedules, continuous administration and repetitive injection. Continuous administration of IFN-alpha to mice decreased the rhythm amplitude of locomotor activity, body temperature, leukocyte counts, and plasma corticosterone levels. The treatment also suppressed the oscillation in the expression of clock genes in the liver. On the other hand, modulation effects were scarcely observed in mice treated with repetitive injection of IFN-alpha. These results indicate that treatment with IFN-alpha does not always modulate the circadian clock function. This notion was also supported by in vitro findings that the inhibitory action of IFN-alpha on the expression of clock genes was dependent on its exposure time to cells. The alteration of clock function induced by IFN-alpha could be avoided by optimizing the dosing schedule.

Circadian Clock in a Mouse Colon Tumor Regulates Intracellular Iron Levels to Promote Tumor Progression

Iron is an important biological catalyst and is critical for DNA synthesis during cell proliferation. Cellular iron uptake is enhanced in tumor cells to support increased DNA synthesis. Circadian variations in DNA synthesis and proliferation have been identified in tumor cells, but their relationship with intracellular iron levels is unclear. In this study, we identified a 24-h rhythm in iron regulatory protein 2 (IRP2) levels in colon-26 tumors implanted in mice. Our findings suggest that IRP2 regulates the 24-h rhythm of transferrin receptor 1 (Tfr1) mRNA expression post-transcriptionally, by binding to RNA stem-loop structures known as iron-response elements. We also found that Irp2 mRNA transcription is promoted by circadian clock genes, including brain and muscle Arnt-like 1 (BMAL1) and the circadian locomotor output cycles kaput (CLOCK) heterodimer. Moreover, growth in colon-26(Δ19) tumors expressing the clock-mutant protein (CLOCKΔ19) was low compared with that in wild-type colon-26 tumor. The time-dependent variation of cellular iron levels, and the proliferation rate in wild-type colon-26 tumor was decreased by CLOCKΔ19 expression. Our findings suggest that circadian organization contributes to tumor cell proliferation by regulating iron metabolism in the tumor.

Alterations of hepatic metabolism in chronic kidney disease via D-box binding protein aggravate the renal dysfunction.

Chronic kidney disease (CKD) is associated with an increase in serum retinol; however, the underlying mechanisms of this disorder are poorly characterized. Here, we found that the alteration of hepatic metabolism induced the accumulation of serum retinol in 5/6 nephrectomy (5/6Nx) mice. The liver is the major organ responsible for retinol metabolism; accordingly, microarray analysis revealed that the hepatic expression of most CYP genes was changed in 5/6Nx mice. In addition, D-box-binding protein (DBP), which controls the expression of several CYP genes, was significantly decreased in these mice. Cyp3a11 and Cyp26a1, encoding key proteins in retinol metabolism, showed the greatest decrease in expression in 5/6Nx mice, a process mediated by the decreased expression of DBP. Furthermore, an increase of plasma transforming growth factor-β1 (TGF-β1) in 5/6Nx mice led to the decreased expression of the Dbp gene. Consistent with these findings, the alterations of retinol metabolism and renal dysfunction in 5/6Nx mice were ameliorated by administration of an anti-TGF-β1 antibody. We also show that the accumulation of serum retinol induced renal apoptosis in 5/6Nx mice fed a normal diet, whereas renal dysfunction was reduced in mice fed a retinol-free diet. These findings indicate that constitutive Dbp expression plays an important role in mediating hepatic dysfunction under CKD. Thus, the aggravation of renal dysfunction in patients with CKD might be prevented by a recovery of hepatic function, potentially through therapies targeting DBP and retinol.

Formation of repressor-inducer-operator ternary complex: negative cooperativity of d-camphor binding to CamR

A repressor composed of homodimeric subunits, as is often found in bacteria, possesses two effector‐binding sites per molecule, enabling sophisticated regulation by the cooperative binding of two effector molecules. Positive cooperativity generates a narrower region of effector concentration for switching, but little is known about the role of negative cooperativity. d‐camphor, an inducer for Pseudomonas putida cytochrome P450cam hydroxylase operon (camDCAB), binds to the homodimeric cam repressor (CamR). Here, we report solid evidence that the complex of CamR and an operator DNA is not dissociated by the first binding of d‐camphor but, at a higher concentration, is dissociated by the second binding. d‐camphor thus binds to the CamR in two steps with negative cooperativity, yielding two distinct dissociation constants of Kd1 = 0.064 ± 0.030 and Kd2 = 14 ± 0.3 μm, as well as the Hill coefficient of 0.56 ± 0.05 (<1). The first binding guarantees the high specificity of the inducer by the high affinity, although the second binding turns on the gene expression at a 200‐fold higher concentration, a more suitable switching point for the catabolism of d‐camphor.