Inhibitory Effect Of Valproic Acid Research Articles

Valproic acid regulates MIEF1 through MST2-HIPPO to suppress breast cancer growth

AimsTo determine the effects of valproic acid (VPA) on anti-proliferative effects and mitochondrial function in breast cancer cells and the underlying mechanisms involved in the effects, with a focus on signal transduction. Main methodsThe inhibitory effect of valproic acid on breast cancer in vivo and in vitro was evaluated by cellular and animal experiments. Mitochondria-related proteins as well as hippo pathway were monitored by western blotting. The effects of VPA on mitochondrial membrane potential, reactive oxygen species, and apoptosis were confirmed by flow cytometry. In addition, the involvement of hippo pathway in the regulation of mitochondrial function by VPA was verified by XMU-MP-1 (MST2 inhibitor). Key findingsIn this study, we highlight that VPA significantly attenuates mitochondrial function, leading to inhibited cell proliferation and reduced colony formation in MCF-7 and MDA-MB-231 breast cancer cells. Mechanistically, VPA-induced suppression of mitochondrial aerobic respiration was mediated by decreased expression of mitochondrial elongation factor 1 through activation of the hippo pathway, resulting in impaired breast cancer growth. In summary, we uncover a novel mechanism of VPA in regulating mitochondrial aerobic respiration, which is essential for developing an effective approach in breast cancer therapy. SignificanceMitochondrial aerobic respiration and its products are the main sources of energy for tumors; therefore, studying the role of mitochondrial function in tumor cells is significant. VPA has been used as a therapeutic agent for cancer. However, the detail mechanism underlying the effects of VPA on mitochondrial function in breast cancer remains unclear.

Valproic acid decreases vascular smooth muscle cell proliferation via protein phosphatase 2A-mediated p70 S6 kinase inhibition

Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Although the abnormal proliferation of vascular smooth muscle cells (VSMCs) is a well-established contributor to the development of various vascular diseases including atherosclerosis, the effect of VPA on VSMC proliferation and its mechanism of action have not been fully revealed. Herein, we investigated the molecular mechanism by which VPA inhibits rat VSMC proliferation. VPA dose-dependently decreased VSMC proliferation, which was accompanied by the dose-dependent decrease in phosphorylation of p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr389), and overexpression of the p70S6K-T389E mutant gene significantly reversed VPA-inhibited VSMC proliferation. Co-treatment with okadaic acid, a specific protein phosphatase 2A (PP2A) inhibitor, significantly restored p-p70S6K-Thr389. Furthermore, knockdown of PP2Ac gene expression by siRNA significantly reversed VPA-inhibited p-p70S6K-Thr389 and VSMC proliferation. Confocal microscopic analyses and co-immunoprecipitation results clearly showed that the physical binding of p70S6K and PP2Ac was promoted by VPA. Valpromide, a VPA's structural derivative with no histone deacetylase (HDAC) inhibition activity, as well as VPA and sodium butyrate, an HDAC inhibitor similar to VPA, decreased VSMC proliferation and p-p70S6K-Thr389, indicating that HDAC is not involved in VPA-inhibited VSMC proliferation. Finally, the inhibitory effects of VPA on p-p70S6K-Thr389 and VSMC proliferation were reiterated in a platelet-derived growth factor (PDGF)-induced in vitro atherosclerosis model. In conclusion, our results demonstrate that VPA decreased cell proliferation via PP2A-mediated inhibition of p-p70S6K-Thr389 in basal and PDGF-stimulated VSMCs. The results suggest that VPA could be used in the treatment and prevention of atherosclerosis and in-stent restenosis.

Valproic Acid Protects Chondrocytes from LPS-Stimulated Damage via Regulating miR-302d-3p/ITGB4 Axis and Mediating the PI3K-AKT Signaling Pathway.

Background: Osteoarthritis (OA) is one of the most common degenerative joint diseases characterized by increased apoptosis and autophagy deficiency. The investigation was performed to examine the effect of valproic acid (VPA) and molecular mechanism related to miR-302d-3p/ITGB4 axis in OA. Methods: The OA clinical samples were obtained from the GEO database to analyze differentially expressed genes. An in vitro OA model was mimicked by LPS in CHON-001 cells. Autophagy-related genes were downloaded from the HADb website, and potential drugs were mined using the CTD website. The upstream factors of ITGB4 were predicted with bioinformatics analysis, which was validated by luciferase activity assay and RIP assay. Cell viability and apoptosis were evaluated using CCK-8 and flow cytometry. The expression levels, including ITGB4, miR-302d-3p, and autophagy-/PI3K-AKT pathway-related markers, were measured by qRT-PCR or/and western blot. Results: Our results showed that miR-302d-3p inhibited cell viability and promoted apoptosis of LPS-treated CHON-001 cells by targeting ITGB4. VPA treatment remarkably alleviated LPS-stimulated injury in CHON-001 cells. The inhibitory effect of VPA on LPS-stimulated damage in CHON-001 cells was weakened by miR-302d-3p overexpression, while it was intensified because of ITGB4 upregulation. Mechanistically, VPA treatment induced a significant decrease in the levels of p-PI3K and p-AKT in LPS-stimulated CHON-001 cells through regulating miR-302d-3p/ITGB4 axis. Conclusion: Overall, VPA treatment may ameliorate LPS-induced injury on chondrocytes via the regulation of miR-302d-3p/ITGB4 pair and the inactivation of the PI3K-AKT pathway.

Inhibition of Porcine Viruses by Different Cell-Targeted Antiviral Drugs.

Antiviral compounds targeting cellular metabolism instead of virus components have become an interesting issue for preventing and controlling the spread of virus infection, either as sole treatment or as a complement of vaccination. Some of these compounds are involved in the control of lipid metabolism and/or membrane rearrangements. Here, we describe the effect of three of these cell-targeting antivirals: lauryl gallate (LG), valproic acid (VPA), and cerulenin (CRL) in the multiplication of viruses causing important porcine diseases. The results confirm the antiviral action in cultured cells of LG against African swine fever virus (ASFV), foot and mouth disease virus (FMDV), vesicular stomatitis virus (VSV), and swine vesicular disease virus (SVDV), as well as the inhibitory effect of VPA and CRL on ASFV infection. Other gallate esters have been also assayed for their inhibition of FMDV growth. The combined action of these antivirals has been also tested in ASFV infections, with some synergistic effects when LG and VPA were co-administered. Regarding the mode of action of the antivirals, experiments on the effect of the time of its addition in infected cell cultures indicated that the inhibition by VPA and CRL occurred at early times after ASFV infection, while LG inhibited a late step in FMDV infection. In all the cases, the presence of the antiviral reduced or abolished the induction of virus-specific proteins. Interestingly, LG also reduced mortality and FMDV load in a mouse model. The possible use of cell-targeted antivirals against porcine diseases is discussed.

Lamotrigine drug interactions in combination therapy and the influence of therapeutic drug monitoring on clinical outcomes in paediatric patients.

The aim was to study the impact of therapeutic drug monitoring (TDM) on paediatric patients on lamotrigine therapy and the evaluation of possible drug interactions, especially in triple antiepileptic drug combinations. During the period of 2001-2015, 1308 pre-dose samples were taken from 430 patients <15years of age as part of routine TDM. Drug interactions were evaluated using calculation of lamotrigine clearance. Valproic acid decreased lamotrigine clearance by 54% in bitherapy, and by 21% in triple therapy with carbamazepine. Carbamazepine increased lamotrigine clearance by 191% in bitherapy. Levetiracetam and topiramate had no effect. The upper limit of lamotrigine therapeutic range (TR) was exceeded in 2% of cases in monotherapy, and in 6%-7% of cases in bi- or triple therapy. About 61% of plasma levels were found within the TR during 2001-2005, compared to 75% and 74% during 2006-2010 and 2011-2015, respectively. Adverse drug reactions (ADRs) were reported in 22 cases. Higher number of supratherapeutic levels in combination therapy led to a 3-fold increase in incidence of ADRs. Seizures occurred more often daily and monthly during 2001-2005 and in patients with three or four antiepileptic drugs in combination. Carbamazepine only partially compensated for the inhibitory effect of valproic acid. Lamotrigine clearance in monotherapy in children is similar to adults, but in polytherapy was found higher susceptibility to induction. A significantly higher number of supratherapeutic lamotrigine levels were found in combinations with valproate. Despite poor correlation with TR, both seizure frequency and ADRs declined after the implementation of TDM.

Valproic acid downregulates heme oxygenase-1 independently of Nrf2 by increasing ubiquitination and proteasomal degradation

AimsHeme oxygenase-1 (HO-1; HMOX1 in human, Hmox1 in mice) is an antioxidative enzyme affecting wide range of sub-cellular processes. It was shown to modulate tumor growth or vascular-related diseases, thus being putative molecular target for tailored therapies. Therefore it is of importance to elucidate novel compounds regulating HO-1 activity/expression and to delineate mechanisms of their action. In the present study we aimed to understand mode of action of valproic acid (VA), an antiepileptic drug, on HO-1 expression. ResultsWe demonstrated that HO-1 expression is decreased by VA at protein but not mRNA level in human alveolar rhabdomyosarcoma cell line CW9019. Nrf2 transcription factor, the activator of HO-1 expression through ARE sequence, was excluded as a mediator of HO-1 decrease, as VA downregulated Bach1, a Nrf2 repressor, concomitantly upregulating ARE activation. Also miRNA-dependent inhibition was excluded as a mechanism of HMOX1 regulation. However, co-immunoprecipitation assay showed a higher level of ubiquitinated HO-1 after VA treatment. Accordingly, MG132, an inhibitor of proteasomal degradation, reversed the effect of VA on HO-1 suggesting that decrease in HO-1 expression by VA is through protein stability.The inhibitory effect of VA on HO-1 was also observed in murine cells including embryonic fibroblasts isolated from Nrf2-deficient mice, what confirms Nrf2-independent effect of the compound. Importantly, VA decreased also HO-1 expression and activity in murine skeletal muscles in vivo. ConclusionOur data indicate that VA downregulates HO-1 by acting through ubiquitin-proteasomal pathway leading to decrease in protein level.

Inhibitory Effects of Valproic Acid in Oxaliplatin-Induced Neuropathy in Rat Model

AIM: Oxaliplatin is a third-generation platinum-based chemotherapy drug, introduced for management of the advanced stages of metastatic colorectal cancer. However, repeated administration of oxaliplatin induced acute and chronic peripheral neuropathy. Valproic acid (VPA) is a neurotherapeutic drug used widespread and worldwide as therapy for seizures, bipolar disorder, and migraine, including children, adult and women of reproductive age. MATERIALS AND METHODS: In the present study, we investigated the effect of VPA in prevention of oxaliplatin-induced periphery neuropathy in the rat model. We demonstrated that VPA (300 mg/kg) relieved the oxaliplatin (4mg/kg)-induced peripheral neuropathy using behavioral tests, biochemical tests, and histopathological and immunohistochemical evaluations. RESULTS: VPA administration significantly attenuated the mechanical hyperalgesia by oxaliplatin-induced in rats. VPA exerted a significant protective effect by reducing the occurrence of multinucleolated neurons and the nucleolar eccentricity caused on lumbar dorsal root ganglion from oxaliplatin-treated rats. It revealed an inhibitory effect of VPA on the number and activation of microglia and astrocytes in the dorsal horn of the spinal cord. However, VPA was unable to prevent demyelination and degeneration of nerve fibers from oxaliplatin-induced peripheral neurotoxicity. COLUSION: The present results demonstrated for the first time that VPA administration ameliorated the oxaliplatin-induced behavioral, biochemical and histopathological changes in rats. The VPA-mediated effects in this study may be attributed to neuroprotection properties and ameliorating oxaliplatin-induced astrocytes and microglial activation. VPA may offer a dual protective approach against etiological factors and resulting maladaptative plasticity.

Induction of Glycosphingolipid GM3 Expression by Valproic Acid Suppresses Cancer Cell Growth

Glycosphingolipid GM3, a known suppressor of epidermal growth factor receptor (EGFR) phosphorylation, inhibits cell proliferation. Valproic acid, conversely, is known as an up-regulator of GM3 synthase gene (ST3GAL5). To test the possibility that valproic acid could inhibit EGFR phosphorylation by increasing the level of GM3 in cells, we treated A431 epidermoid carcinoma cells with valproic acid and found that valproic acid treatment caused an about 6-fold increase in the GM3 level but only a marginal increase in the GM2 level in these cells and that the observed increase in GM3 level was valproic acid dose-dependent. Consistent with this observation, valproic acid treatment induced GM3 synthase gene expression by about 8-fold. Furthermore, phosphorylation of EGFR was reduced, and cell proliferation was inhibited following valproic acid treatment. Consistent with these results, transient expression of GM3 synthase gene in A431 cells also increased cellular level of GM3, reduced phosphorylation of EGFR, and inhibited cell proliferation. Treatment with l-phenyl-2-decanoylamino-3-morpholino-l-propanol, an inhibitor of glucosylceramide synthesis, decreased the cellular level of GM3 and reduced the inhibitory effects of valproic acid on EGFR phosphorylation and cell proliferation. These results suggested that induction of GM3 synthesis was enough to inhibit proliferation of cancer cells by suppressing EGFR activity. Valproic acid treatment similarly increased the GM3 level and reduced phosphorylation of EGFR in U87MG glioma cells and inhibited their proliferation. These results suggested that up-regulators of GM3 synthase gene, such as valproic acid, are potential suppressors of cancer cell proliferation.

Valproic acid inhibits proliferation of HER2-expressing breast cancer cells by inducing cell cycle arrest and apoptosis through Hsp70 acetylation.

Breast cancer encompasses a heterogeneous group of diseases at the molecular level. It is known that chemo-sensitivity of breast cancer depends on its molecular subtype. We investigated the growth inhibitory effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, and the mechanism of this inhibition on four breast cancer cell lines with different molecular subtypes. The growth inhibitory effect of VPA in the four different breast cancer cell lines was investigated. The alteration of levels of p21 WAF1, cleaved caspase-3, acetylated Heat shock protein (Hsp) 90, acetylated Hsp70, and acetylated α-tubulin by VPA was examined in VPA-sensitive, human epidermal receptor 2 (HER2)-overexpressing SKBR3 cells. The cell growth inhibition of breast cancer cell lines was dependent on the dose and exposure time of VPA. The cell growth of HER2-overexpressing SKBR3 cell line was inhibited by VPA to a much greater degree than other cell lines studied. In SKBR3 cell line, VPA upregulated expression of p21 WAF1 and cleaved caspase-3 in the early phase. VPA markedly increased Hsp70 acetylation in a time-dependent manner but did not increase Hsp90 acetylation. Our data demonstrated that VPA inhibited cell proliferation and induced cell cycle arrest and apoptosis of HER2-overexpressing breast cancer cells. This anti-proliferation effect might be the direct function of VPA as an HDAC inhibitor. We propose an alternative mechanism whereby acetylation of Hsp70 disrupts the function of Hsp90 and leads to downregulation of its client proteins, including HER2 that might be the indirect function of VPA, in the sense that non-histone proteins are acetylated.

Inhibitory effects of valproic acid in DNA double-strand break repair after irradiation in esophageal squamous carcinoma cells.

Radiation therapy is one of the most promising therapeutic strategies in unresectable esophageal squamous cell carcinoma (ESCC). The histone deacetylase (HDAC) inhibitor has been shown to enhance radiosensitivity. Valproic acid (VPA) is a well-known drug used to treat seizure disorders and epilepsy, and has been shown to inhibit HDACs. We recently reported that a clinically safe dose of VPA enhances radiation‑induced cytotoxicity in human ESCC cells. However, the mechanism of radiosensitizing effect of VPA has not yet been confirmed. The present study examined the effect of VPA on DNA double-strand break (DSB) repair after radiation in the human ESCC cell lines KES, TE9 and TE11 by examining H2AX phosphorylation (γH2AX) levels as a marker of radiation‑induced DSBs. The present study also examined whether VPA inhibited radiation-induced DNA DSB repair by suppressing non-homologous end joining (NHEJ), focusing particularly on the acetylation of Ku70. VPA was shown to prolong γH2AX levels after irradiation in all three ESCC cell lines. Moreover, prolonged γH2AX foci formation after irradiation was also observed by immunocytochemistry following VPA pretreatment in KES and TE9 cells. VPA was shown to induce Ku70 acetylation after irradiation in all three ESCC cell lines. Our results suggest that VPA prolonged radiation‑induced DSBs by inhibiting NHEJ in DSB repair pathways in ESCC. VPA could therefore be used as an effective radiosensitizer in ESCC radiotherapy.

A novel mechanism for inhibition of lipopolysaccharide-induced proinflammatory cytokine production by valproic acid

The inhibitory effect of valproic acid (VPA) on lipopolysaccharide (LPS)-induced inflammatory response was studied by using mouse RAW 264.7 macrophage-like cells. VPA pretreatment attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen-activated protein kinases. VPA reduced phosphorylation of MDM2, an ubiquitin ligase and then prevented LPS-induced p53 degradation, followed by enhanced p53 expression. Moreover, p53 small interfering RNA (siRNA) abolished the inhibitory action of VPA on LPS-induced NF-κB p65 transcriptional activation and further LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. VPA prevented LPS-induced degradation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and up-regulated the PTEN expression. Taken together, VPA was suggested to down-regulate LPS-induced NF-κB-dependent transcriptional activity via impaired PI3K/Akt/MDM2 activation and enhanced p53 expression. A detailed mechanism for inhibition of LPS-induced inflammatory response by VPA is discussed.

Inhibitory effect of valproic acid on bladder cancer in combination with chemotherapeutic agents in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) are a promising class of drugs that act as antiproliferative agents by promoting differentiation and inducing apoptosis. Valproic acid (VPA) is an HDACI that has been widely used as an anti-convulsant and shows promise as a chemotherapeutic drug for a number of tumor cells. The present study aimed to investigate the inhibitory effect of VPA on the viability of bladder cancer cells and its synergistic effect with chemotherapeutic agents in vitro and in vivo. The cell viability of human bladder cancer cell lines following treatment with VPA and/or VPA in combination with mitomycin C, cisplatin (DDP) and adriamycin were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining was used to observe the morphology of the apoptotic cells. Survivin protein and acetylated histone H3 levels were quantified using western blot analysis. The in vivo tumor growth inhibition of VPA was determined in rats with N-methyl-N-nitrosourea-induced bladder cancer. VPA significantly inhibited the growth of the bladder cancer cells in a concentration- and time-dependent manner. Furthermore, improved results were achieved for tumor inhibition when VPA was combined with chemotherapeutic agents in vitro and in vivo. Survivin expression decreased and acetylated histone H3 expression increased in the bladder cancer cells following the treatment with VPA. Intravesical injections of VPA were able to inhibit tumor progression when combined with DDP. In conclusion, VPA acts as an HDACI that has a direct anticancer effect and markedly enhances the action of several chemotherapy agents. VPA may sensitize bladder cancer to anticancer drugs by downregulating survivin expression.

Abstract 1004: Histone deacetylase inhibitors: Potential epigenetic therapeutic drugs for cholangiocarcinoma.

Abstract Cholangiocarcinoma (CCA), a cancer of biliary epithelium, is a rare tumor with poor prognosis which has been increasing markedly worldwide in the past decade. The surgical resection remains a potentially curative treatment of CCA. Due to the late clinical presentation, a small number of patients can undergo for surgery. Adjuvant therapy such as chemotherapy and radiotherapy has been used for CCA. Although various chemotherapeutic agents e.g. 5-fluorouracil (5-FU), gemcitabine, and cisplatin, either alone or in combination, have been widely used in CCA, the response rate is very poor with a partial response approximately 10-20%. Therefore, new agents and innovative approaches of therapy are important subjects focusing in effective treatment of CCA. Histone deacetylases (HDACs) mediate deacetylation of acetylated histones resulting in chromatin condensation and transcriptional repression. HDAC inhibitors (HDACIs) inhibit the deacetylation of histones and weaken the histone-DNA interactions, thereby permitting a more relaxed conformation of chromatin which contributes to increasing gene transcription and enabling to induce differentiation, cell cycle arrest, and apoptosis of tumor cells. Recently, HDACIs are emerging as an exciting new class of potential anticancer agents for the treatment of solid and hematological malignancies with relatively resistant in normal cells. According to chemical structures, HDACIs can be classified into several groups such as sodium butyrate, valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA). The authors aimed to investigate the mRNA expression of HDACs1-10 in a panel of CCA derived cell lines (M213, M214 and KKU-100) and an immortal biliary cell line (MMNK1) using semi-quantitative reverse transcription-polymerase chain reaction. The inhibitory effect of VPA and SAHA, either as a single agent or combination with classical chemotherapeutic drug, 5-FU on cell proliferation was determined by sulforhodamine B assay. The result showed that HDACs 1-10 were expressed in all cell types studied suggesting that histone deacetylation is one of epigenetic mechanisms mediating aberrantly silenced genes in CCA. Both VPA and SAHA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 which was less sensitive to 5-FU showed highly sensitive to both VPA and SAHA. Simultaneously combined sub-toxic doses of HDACIs and 5-FU significantly inhibited cell proliferation in CCA cell lines compared to single agent treatment (P ≤ 0.01), while sequentially combined treatments exhibited lower responsiveness. This study suggests the prospective administration of HDACIs as potential epigenetic therapeutic drugs for CCA treatment efficacy. Citation Format: Temduang Limpaiboon, Ruethairat Sriraksa. Histone deacetylase inhibitors: Potential epigenetic therapeutic drugs for cholangiocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1004. doi:10.1158/1538-7445.AM2013-1004

Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line

BackgroundManagement of peritoneal dissemination is the most critical problem in gastric cancer. This study was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA.MethodsThe effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay.ResultsOCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl-α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001).ConclusionsVPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer.

Inhibitory effect of valproic acid on ovarian androgen biosynthesis in rat theca-interstitial cells

The objective of the study was to evaluate the effect of valproic acid (VPA) on ovarian androgen biosynthesis in primary cultures of theca-interstitial (T-I) cells isolated from rat ovaries. Ovarian T-I cells were cultured with VPA in the presence or absence of hCG. VPA did not increase basal or hCG-stimulated androgen synthesis when added to primary cultures of T-I cells. However, the addition of VPA caused a marked concentration-dependent inhibitory effect on hCG-stimulated androstendione synthesis. Treatment of T-I cells with 8-Bromo-cAMP resulted in a marked increase in the production of androstenedione, and VPA inhibited this stimulatory effect, suggesting that the mechanism of VPA's inhibitory effect on androstenedione production occurs at a step after second messenger activation. Treatment of T-I cells with hCG resulted in a significant increase in the mRNA expression of steroidogenic enzymes CYP17A1 and 17β-hydroxysteroid dehydrogenase. Addition of VPA sharply blunted the stimulatory effect of hCG, reducing the mRNA expression of the steroidogenic enzymes to basal levels. In conclusion, VPA exerts an inhibitory effect on hCG-stimulated androgen synthesis in rat T-I cells.

Inhibitory Effect of Valproic Acid on Cytochrome P450 2C9 Activity in Epilepsy Patients

Drug interactions constitute a major problem in the treatment of epilepsy because drug combinations are so common. Valproic acid is a widely used anticonvulsant drug with a broad therapeutic spectrum. Case reports suggest interaction between valproic acid and other drugs metabolized mainly by cytochrome P450 isoforms. The aim of this study was to evaluate the inhibitory effect of valproic acid on cytochrome P450 2C9 (CYP2C9) activity by using losartan oxidation as a probe in epilepsy patients. Patients were prescribed sodium valproate (mean 200 mg/day for the first week and 400 mg/day in the following period) according to their clinical need. A single oral dose of 25 mg losartan was given to patients before and after the first dose, first week and 4 weeks of valproic acid treatment. Losartan and E3174, the CYP2C9-derived carboxylic acid metabolite of losartan in 8 hr urine were assayed by using high pressure liquid chromatography. Urinary losartan/E3174 ratio did not change significantly on the first day (0.9, 0.3-3.5; median, range), and first week (0.6, 0.2-3.8; median, range), while a significant increase was observed after 4 weeks of valproic acid treatment (1.1, 0.3-5.7; median, range) as compared to that of measured before valproic acid administration (0.6, 0.1-2.1; median, range) (P = 0.039). The degree of inhibition was correlated with the steady-state plasma concentrations of valproic acid (r(2) = 0.70, P = 0.04). The results suggest an inhibitory effect of valproic acid on CYP2C9 enzyme activity in epilepsy patients at steady state. The risk of pharmacokinetic drug-drug interactions should be taken into account during concomitant use of valproic acid and CYP2C9 substrates.

Inhibitory effect of valproic acid on cytochrome P450 2C9 activity in epilepsy patients

Drug interactions constitute a major problem in the treatment of epilepsy because drug combinations are common. Case reports suggest an interaction between valproic acid and drug substrates of CYP2C9. The aim of this study was to evaluate the inhibitory effect of valproic acid on CYP2C9 activity by using losartan oxidation as a marker in epilepsy patients. A single oral dose of 25 mg losartan was given to patients (n=11) at the baseline. Losartan and its CYP2C9 derived metabolite E3174 were assayed in 8-hour urine samples by using HPLC. The same procedure was repeated after the first dose and at the completion of the first and fourth weeks of valproic acid treatment. Urinary losartan/E3174 ratio (median, range) did not change significantly at the first day or the first week, while a significant increase was observed at the fourth week of valproic acid treatment (1.09, 0.28–5.71) as compared with the baseline (0.60, 0.10–2.07) (P=0.039). The degree of inhibition was correlated with the steady state plasma concentrations of valproic acid (r2=0.82, P=0.018). These results provide an in vivo evidence that valproic acid is an inhibitor of CYP2C9 at concentrations reached during steady state in human. The risk of pharmacokinetic drug-drug interactions should be considered during concomitant use of valproic acid and CYP2C9 substrates. (Supported by TUBITAK-SBAG COST B25-105S027 and UY/TUBA-GEBIP/2005-17)

Valproic Acid Inhibits Invasiveness in Bladder Cancer but Not in Prostate Cancer Cells

Histone deacetylase inhibitors (HDACIs) represent a promising new class of antineoplastic agents that affect proliferation, differentiation, and apoptosis in both solid and hematologic malignancies. In addition, HDACIs can alter the expression of at least one cellular adhesion molecule, the coxsackie and adenovirus receptor, in bladder cancer. Because HDACIs can increase expression of a known cellular adhesion molecule, we hypothesized that migration and/or invasion may also be affected. We evaluated this hypothesis using valproic acid (VPA), a commonly prescribed anticonvulsant recently shown to have potent HDACI activity, in the bladder cancer cell lines T24 TCC-SUP, HT1376, and RT4. Analyses of cell migration and invasion were both qualitative (fluorescent microscopy) and quantitative (static and dynamic migration/invasion assays). Our results show that acute VPA treatment (72 h) causes a dose-dependent decrease in invasion for all bladder cancer cell lines, except RT4, a noninvasive papilloma. Migration, in contrast, was not affected by VPA treatment. The inhibitory effect of VPA may be cancer type-specific, because there was no difference in invasion between treated and untreated prostate cancer cell lines LNCaP, PC3, and DU145. Furthermore, when administered chronically (34 days), VPA significantly inhibits growth of T24t tumor xenografts. Our data suggest that VPA exerts some of its antineoplastic effects by inhibiting invasion as well as tumor growth, and thus it may represent a novel adjuvant strategy for patients at high risk of recurrence and/or progression of muscle invasive bladder cancer.

Valproic acid inhibits substance P-induced activation of protein kinase C epsilon and expression of the substance P receptor.

The neuropeptide substance P (SP) has been hypothesized to be involved in the etiopathology of affective disorders. This hypothesis is based on the findings that neurokinin-1-receptor antagonists have antidepressant effects in depressed patients and that SP may worsen mood. In this study, we investigated the effect of the mood-stabilizing agents valproic acid (VPA), carbamazepine, and lithium on SP-induced gene expression. As a model system, we used primary rat astrocytes and human astrocytoma cells, which both express functional SP-receptors and, upon stimulation with SP, synthesize interleukin-6 (IL-6), a cytokine which has been shown to be elevated during the acute depressive state. We found that VPA dose-dependently inhibited SP-induced IL-6 synthesis which was seen with pre-incubation periods of 30 min, 3, 7 and 14 days, whereas carbamazepine and lithium showed no inhibitory effect. The inhibitory effect of VPA was not mediated by inhibition of the stress-regulated kinases p38 and p42/44 (Erk1/2) but by inhibition of protein kinase C epsilon activation. Furthermore, VPA down-regulated the expression of the substance P receptor (neurokinin(NK)-1-receptor) as assessed by real-time PCR. Whether both mechanisms contribute to the mood-stabilizing properties of VPA has to be evaluated in further studies.

Inhibitory effect of valproate on weak organic acid uptake in rat renal proximal tubules

The effects of an antiepileptic drug, valproic acid (VPA), on transport mechanisms involved in renal excretion of anionic xenobiotics were investigated on rat renal proximal tubules in vitro. It was found that VPA (0.1–1 mM) dose dependently inhibited the baseline uptake of a marker organic anion, fluorescein, in the tubules. The inhibition could not be exclusively accounted for by competition between VPA and fluorescein. Taking into account a proposed relationship between the weak organic anion uptake and ammoniagenesis, the influence of VPA (0.5 mM) on the effects of glutamine and glutamate (both at 5 mM) on fluorescein uptake and ammonia production were examined. Glutamine stimulated ammonia production by the tubules, with the glutamine-induced ammoniagenesis being further augmented by VPA, while glutamate failed to affect the basal ammoniagenesis. Both glutamine (5 mM) and glutamate (5 mM) slightly inhibited fluorescein uptake, with the inhibitory effects not modified by VPA. Thus, there was no coincidence in the effects of VPA on organic anion uptake and renal ammoniagenesis. At the same time, the inhibitory effect of VPA (0.5 mM) on fluorescein uptake was largely overcome by addition of pyruvate (5 mM) to the incubation medium. In addition, VPA strongly inhibited glucose production from pyruvate. A known modulator of pyruvate metabolism, dichloroacetic acid (DCA, 1 mM), also inhibited fluorescein uptake, although its inhibitory effect was less pronounced than that of VPA. Both inhibitors failed to alter the tissue content of α-ketoglutarate or lactate but did slightly augment the pyruvate level. The inhibitory effects of VPA and DCA on the baseline fluorescein uptake were not additive, suggesting their similar intracellular targeting. It is assumed that the inhibitory effect of VPA on baseline fluorescein uptake in rat renal proximal tubules in vitro may be associated with its action on pyruvate metabolism.