Vitamin K3 (Menadione) is a multifunctional microbicide acting as a photosensitizer and synergizing with blue light to kill drug-resistant bacteria in biofilms.

Cutaneous bacterial wound infections typically involve gram-positive cocci such as Staphylococcus aureus (SA) and usually become biofilm infections. Bacteria in biofilms may be 100-1000-fold more resistant to an antibiotic than the clinical laboratory minimal inhibitory concentration (MIC) for that antibiotic, contributing to antimicrobial resistance (AMR). AMR is a growing global threat to humanity. One pathogen-antibiotic resistant combination, methicillin-resistant SA (MRSA) caused more deaths globally than any other such combination in a recent worldwide statistical review. Many wound infections are accessible to light. Antimicrobial phototherapy, and particularly antimicrobial blue light therapy (aBL) is an innovative non-antibiotic approach often overlooked as a possible alternative or adjunctive therapy to reduce antibiotic use. We therefore focused on aBL treatment of biofilm infections, especially MRSA, focusing on in vitro and ex vivo porcine skin models of bacterial biofilm infections. Since aBL is microbicidal through the generation of reactive oxygen species (ROS), we hypothesized that menadione (Vitamin K3), a multifunctional ROS generator, might enhance aBL. Our studies suggest that menadione can synergize with aBL to increase both ROS and microbicidal effects, acting as a photosensitizer as well as an ROS recycler in the treatment of biofilm infections. Vitamin K3/menadione has been given orally and intravenously worldwide to thousands of patients. We conclude that menadione/Vitamin K3 can be used as an adjunct to antimicrobial blue light therapy, increasing the effectiveness of this modality in the treatment of biofilm infections, thereby presenting a potential alternative to antibiotic therapy, to which biofilm infections are so resistant.

Disruption of multiple copies of the Prostaglandin F2alpha synthase gene affects oxidative stress response and infectivity in Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a serious chronic parasitic disease, currently treated with Nifurtimox (NFX) and Benznidazole (BZ). In addition to high toxicity, these drugs have low healing efficacy, especially in the chronic phase of the disease. The existence of drug-resistant T. cruzi strains and the occurrence of cross-resistance between BZ and NFX have also been described. In this context, it is urgent to study the metabolism of these drugs in T. cruzi, to better understand the mechanisms of resistance. Prostaglandin F2α synthase (PGFS) is an enzyme that has been correlated with parasite resistance to BZ, but the mechanism by which resistance occurs is still unclear. Our results show that the genome of the CL Brener clone of T. cruzi, contains five PGFS sequences and three potential pseudogenes. Using CRISPR/Cas9 we generated knockout cell lines in which all PGFS sequences were disrupted, as shown by PCR and western blotting analyses. The PGFS deletion did not alter the growth of the parasites or their susceptibility to BZ and NFX when compared to wild-type (WT) parasites. Interestingly, NTR-1 transcripts were shown to be upregulated in ΔPGFS mutants. Furthermore, the ΔPGFS parasites were 1.6 to 1.7-fold less tolerant to oxidative stress generated by menadione, presented lower levels of lipid bodies than the control parasites during the stationary phase, and were less infective than control parasites.

Vitamin K: A novel cancer chemosensitizer.

Cancer incidences are growing rapidly and causing millions of deaths globally. Cancer treatment is one of the most exigent challenges. Drug resistance is a natural phenomenon and is considered one of the major obstacles in the successful treatment of cancer by chemotherapy. Combination therapy by the amalgamation of various anticancer drugs has suggested modulating tumor response by targeting various signaling pathways in a synergistic or additive manner. Vitamin K is an essential nutrient and has recently been investigated as a potential anticancer agent. The combination of vitamin K analogs, such as vitamins K1, K2, K3, and K5, with other chemotherapeutic drugs have demonstrated a safe, cost-effective, and most efficient way to overcome drug resistance and improved the outcomes of prevailing chemotherapy. Published reports have shown that vitamin K in combination therapy improved the efficacy of clinical drugs by promoting apoptosis and cell cycle arrest and overcoming drug resistance by inhibiting P-glycoprotein. In this review, we discuss the mechanism, cellular targets, and possible ways to develop vitamin K subtypes into effective cancer chemosensitizers. Finally, this review will provide a scientific basis for exploiting vitamin K as a potential agent to improve the efficacy of chemotherapeutic drugs.

Apoptotic Effects of a Thioether Analog of Vitamin K3 in a Human Leukemia Cell Line.

Research suggests that thioether analogs of vitamin K3 (VK3) can act to preserve the phosphorylation of epidermal growth factor receptors by blocking enzymes (phosphatases) responsible for their dephosphorylation. Additionally, these derivatives can induce apoptosis via mitogen-activated protein kinase and caspase-3 activation, inducing reactive oxygen species (ROS) production, and apoptosis. However, vitamin K1 exhibits only weak inhibition of phosphatase activity, while the ability of VK3 to cause oxidative DNA damage has raised concerns about carcinogenicity. Hence, in the current study, we designed, synthesized, and screened a number of VK3 analogs for their ability to enhance phosphorylation activity, without inducing off-target effects, such as DNA damage. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay revealed that each analog produced a different level of cytotoxicity in the Jurkat human leukemia cell line; however, none elicited a cytotoxic effect that differed significantly from that of the control. Of the VK3 analogs, CPD5 exhibited the lowest EC50, and flow cytometry results showed that apoptosis was induced at final concentrations of ≥10 µM; hence, only 0.1, 1, and 10 µM were evaluated in subsequent assays. Furthermore, CPD5 did not cause vitamin K-attributed ROS generation and was found to be associated with a significant increase in caspase 3 expression, indicating that, of the synthesized thioether VK3 analogs, CPD5 was a more potent inducer of apoptosis than VK3. Hence, further elucidation of the apoptosis-inducing effect of CPD5 may reveal its efficacy in other neoplastic cells and its potential as a medication.

Vitamin K3 chloro derivative (VKT-2) inhibits HDAC6, activates autophagy and apoptosis, and inhibits aggresome formation in hepatocellular carcinoma cells.

Epigenetics plays a vital role in regulating gene expression and determining the specific phenotypes of eukaryotic cells. Histone deacetylases (HDACs) are important epigenetic regulatory proteins effecting multiple biological functions. Particularly, HDAC6 has become a promising anti-cancer drug target because of its regulation of cell mobility, protein trafficking, degradation of misfolded proteins, cell growth, apoptosis, and metastasis. In this study, we identified one out of six vitamin K3 derivatives, VKT-2, as HDAC6 inhibitor using molecular docking and cell viability assays in HDAC6-overexpressing HuH-7 cancer cells. Microscale thermophoresis and HDAC6 enzymatic assays revealed that VKT-2 bound to HDAC6 and inhibited its function. We further identified its cytotoxic activity. VKT-2 hyperacetylated HDAC6 substrates and disturbed tubulin integrity leading to significant inhibition of tumor migration in both HuH-7 spheroids and U2OS-GFP-α-tubulin cells. Moreover, VKT-2 induced autophagic and apoptotic cell death in HuH-7, while aggresome formation was restrained after VKT-2 treatment. A HuH-7 cell-xenograft model in zebrafish larvae provided evidence that VKT-2 inhibited the tumor growth in vivo. To best of our knowledge, it is the first time to demonstrate that vitamin k3 derivatives (VKT-2) inhibits HDAC6 in solid tumor cells. These unique findings suggested that VKT-2 is a promising anti-cancer agent targeting HDAC6.

Improved redox anti-cancer treatment efficacy through reactive species rhythm manipulation.

Rhythms in the pseudo-steady state (PSS) levels of reactive species (RS), particularly superoxide and hydroxyl radicals, exist in cancer cells. The RS rhythm characteristics, particularly frequency and amplitude, are entrained (reset) by the anticancer compounds/drugs. In this work, we show for the first time that the phase of the RS rhythm at which the drug is added is significantly important in determining the cytotoxicity of anticancer compounds/drugs such as menadione and curcumin, in two different cancer cell lines. Curcumin, the more effective of the two drugs (IC50 = 15 µM, SiHa; 6 µM, HCT116) induced reset of superoxide and hydroxyl rhythms from 15.4 h to 9 h, and 25 h to 11 h respectively, as well as caused increases in these radical levels. However, menadione (IC50 = 20 µM, SiHa; 17 µM, HCT116) affected only the superoxide levels. Drug treatment at different time points/phase of the RS rhythm resulted in a maximum of 27% increase in cytotoxicity, which is significant. Further, we report for the first time, an unexpected absence of a correlation between the intracellular PSS RS and antioxidant levels; thus, the practice of using antioxidant enzyme levels as surrogate markers of intracellular oxidative stress levels may need a re-consideration. Therefore, the RS rhythm could be a fundamental/generic target to manipulate for improved cancer therapy.

Comparison of the Effect of Native 1,4-Naphthoquinones Plumbagin, Menadione, and Lawsone on Viability, Redox Status, and Mitochondrial Functions of C6 Glioblastoma Cells.

BACKGROUND: 1,4-naphthoquinones, especially juglone, are known for their anticancer activity. However, plumbagin, lawsone, and menadione have been less investigated for these properties. Therefore, we aimed to determine the effects of plumbagin, lawsone, and menadione on C6 glioblastoma cell viability, ROS production, and mitochondrial function. METHODS: Cell viability was assessed spectrophotometrically using metabolic activity method, and by fluorescent Hoechst/propidium iodide nuclear staining. ROS generation was measured fluorometrically using DCFH-DA. Oxygen uptake rates were recorded by the high-resolution respirometer Oxygraph-2k. RESULTS: Plumbagin and menadione displayed highly cytotoxic activity on C6 cells (IC50 is 7.7 ± 0.28 µM and 9.6 ± 0.75 µM, respectively) and caused cell death by necrosis. Additionally, they increased the amount of intracellular ROS in a concentration-dependent manner. Moreover, even at very small concentrations (1-3 µM), these compounds significantly uncoupled mitochondrial oxidation from phosphorylation impairing energy production in cells. Lawsone had significantly lower viability decreasing and mitochondria-uncoupling effect, and exerted strong antioxidant activity. CONCLUSIONS: Plumbagin and menadione exhibit strong prooxidant, mitochondrial oxidative phosphorylation uncoupling and cytotoxic activity. In contrast, lawsone demonstrates a moderate effect on C6 cell viability and mitochondrial functions, and possesses strong antioxidant properties.

Therapeutic role of calcium and vitamin K3 in chemically induced hepatocarcinogenesis - new tools for cancer treatment.

HCC has been reported to be immensely occurring carcinoma worldwide. Recent days the mortality occurred due to liver cancer has also been found to be increased at an alarming speed affecting mostly the young patients. The aim of the current study was to decipher the role of calcium and vitamin K3 in the treatment of chemically induced hepatocarcinogenesis in the male Wistar rats. Liver cancer was induced via a subnecrogenic dose of 160 mg/kg body weight, diethylnitrosamine (DENA) when associated with fasting/refeeding in male Wistar rats. It elevated the serum glutamate oxaloacetate (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), bilirubin, total cholesterol (CH), triglycerides (TG), alfa-fetoprotein (AFP) and reduced high-density lipoprotein (HDL). Histopathological examination of liver tissue showed marked carcinogenicity of the chemical carcinogen. Food, water intake and animal weights were also assessed, respectively. The animals exposed to DENA showed a significant decrease in the body weight. The elevated levels of serum SGOT, SGPT, ALP, AFP, TC and TG were restored by administration of calcium and Vit K (ad libitum) combination at higher dose than the normal dietary requirement (3 mg/kg) daily for 12 weeks p.o. Physiological and biochemical analysis showed the beneficial effects of calcium and vitamin K3 combination in the animals exposed to DENA. The results deciphered the beneficial effects of calcium and vitamin K3 in combination.

Endogenous carbon monoxide production by menadione.

INTRODUCTION: Smokers have a significantly decreased risk of pre-eclampsia (PE), possibly attributed to an increase in blood carbon monoxide (CO) concentrations. At physiological concentrations, CO has been demonstrated to have placental vasodilatory and anti-inflammatory properties. Increasing endogenous CO production may have therapeutic potential to either prevent or treat PE. Menadione (MD), synthetic vitamin K3, increases CO in rat microsomes. Our objective was to investigate MD's ability to increase endogenous CO concentrations in pregnancy. METHODS: Three experiments were completed. First, in vitro CO production was measured using isolated GD15 placentas. Second, non-pregnant normotensive mice received no, 1.5, 4.0 or 6.5 g/L MD for 7 days. Lastly, pregnant normotensive mice received either no or 6.5 g/L MD in water from GD10.5 to GD17.5. Consumption was measured as average daily water intake per gram of body weight. Maternal and fetal CO levels in the blood and tissue were quantified using headspace gas chromatography. RESULTS: MD significantly increased CO production in isolated GD15 placentas. In both pregnant and non-pregnant experiments, splenic CO, hepatic CO, and splenic mass were higher in treated mice compared to controls (all p < 0.05). Maternal %COHb and Hb in treated dams were not significantly different compared to controls. The fetal:placental mass ratio was significantly lower in the treatment group (p = 0.002). DISCUSSION: Placental CO production was observed in GD15 placentas after co-incubation with MD. MD administration increased CO in the liver and spleens of pregnant mice. Further investigation into different doses of MD is required to identify one without demonstrable fetal/placental effects.

Cancer-specific chemotherapeutic strategy based on the vitamin K3 mediated ROS regenerative feedback and visualized drug release in vivo.

A promising theranostic nanosystem VK3-CPT@Ru-CD is designed and fabricated by the host-guest driven self-assembly between the fluorescent adamantine-functionalized Ru(II) complexes and the ROS-labile-cyclodextrin modified thioketal linkers, in which anticancer drug camptothecin (CPT) and vitamin K3 (VK3) are effectively co-encapsulated. On account of the generative feedback between the intracellular redox cycling of VK3 and the high degree of ROS-triggered collapse of nanoparticles, VK3-CPT@Ru-CD can facilitate cancer-specific ROS amplification and drug release selectively in cancer cells, thus realizing the selective killing of tumor with minimal side-effects both in vitro and in vivo, the therapeutic effect of which is more prominent than the free anti-cancer drugs. More interestingly, the menadione structure of encapsulated VK3 can effectively quench the inherent fluorescence of Ru-CD, and a fluorescence lightening up phenomenon is observed accompanied with the ROS-triggered drug release, which can be utilized for real-time tracking of drug release in vitro and in vivo.

Evaluation of inhibitory effect of redox-active antimalarial drug against Babesia microti in mice

Babesiosis is an emerging, tick-transmitted disease caused by the intraerythrocytic parasite Babesia microti. In immunocompetent individuals, B. microti infection quickly resolves after antibabesial treatment. Immunocompromised patients and those of advanced age experience chronic and relapsing babesiosis, accompanied by severe complications and often, a fatal outcome. In these individuals, B. microti infection may persist despite multiple courses of treatment with antiprotozoal drugs. The increasing incidence of human babesiosis caused by B. microti, coupled with a growing number of immunosuppressed people who do not respond to standard antibabesial therapy, emphasises the need for new therapeutics for this protozoan infection with more effective mechanisms of action. Plasmodione, namely 3-[4-(trifluoromethyl)benzyl]-menadione, acts as a redox cycler and disrupts the redox homeostasis of Plasmodium-infected erythrocytes. The present study was designed to evaluate the potential inhibitory effect of this novel antimalarial compound against intraerythrocytic stages of B. microti in mice. Our results demonstrate that plasmodione did not reduce the level of parasitemia in B. microti-infected mice, indicating that interfering with the parasite redox balance is not an effective strategy to restrict the division of this protozoan. The mechanism of parasite resistance to plasmodione may be based on the differences in the oxidative metabolisms of Babesia and Plasmodium parasites inside infected erythrocytes. The significance of our results is discussed in relation to the development of novel antibabesial drugs based on redox-active benzylmenadiones.

Combined calcitriol and menadione reduces experimental murine triple negative breast tumor.

BACKGROUND: Calcitriol (D) or 1,25(OH)2D3 inhibits the growth of several tumor cells including breast cancer cells, by activating cell death pathways. Menadione (MEN), a glutathione-depleting compound, may be used to potentiate the antiproliferative actions of D on cancer cells. We have previously shown in vitro that MEN improved D-induced growth arrest on breast cancer cell lines, inducing oxidative stress and DNA damage via ROS generation. Treatment with MEN+D resulted more effective than D or MEN alone. OBJECTIVE: To study the in vivo effect of calcitriol, MEN or their combination on the development of murine transplantable triple negative breast tumor M-406 in its syngeneic host. METHODS: Tumor M-406 was inoculated s.c., and when tumors reached the desired size, animals were randomly assigned to one of four groups receiving daily i.p. injections of either sterile saline solution (controls, C), MEN, D, or both (MEN+D). Body weight and tumor volume were recorded three times a week. Serum calcium was determined before and at the end of the treatment, at which time tumor samples were obtained for histological examination. RESULTS: None of the drugs, alone or in combination, affected mice body weight in the period studied. The combined treatment reduced tumor growth rate (C vs. MEN+D, P<0.05) and the corresponding histological sections exhibited small remaining areas of viable tumor only in the periphery. A concomitant DNA fragmentation was observed in all treated groups and MEN potentiated the calcitriol effect on tumor growth. CONCLUSIONS: As previously observed in vitro, treatment with MEN and D delayed tumor growth in vivo more efficiently than the individual drugs, with evident signals of apoptosis induction. Our results propose an alternative protocol to treat triple negative breast cancer, using GSH depleting drugs together with calcitriol, which would allow lower doses of the steroid to maintain the antitumor effect while diminishing its adverse pharmacological effects.

Placebo-controlled phase II study of vitamin K3 cream for the treatment of cetuximab-induced rash.

PURPOSE: Cetuximab inhibits the epidermal growth factor receptor (EGFR), and papulopustular eruptions is a frequent side effect. Vitamin K3 (menadione) has preclinically shown to be a potential activator of the EGFR by phosphorylating the receptor (pEGFR). The present randomised study investigated the effect of a vitamin K3 cream on cetuximab-induced rash. MATERIALS AND METHODS: Thirty patients were included in this double-blinded placebo-controlled trial. Patients receiving cetuximab 500 mg/m2 every second week plus chemotherapy for metastatic cancer were included. In each patient, vitamin K3 cream and placebo were applied twice daily on two separate areas of the skin of minimum 10 × 10 cm for up to 2 months. Papulopustular eruptions were evaluated clinically and monitored by clinical photos. Skin biopsies, from ten patients taken before and after 1 month of treatment from each treatment area, were stained for EGFR and pEGFR. RESULTS: Application of vitamin K3 cream twice daily during treatment with cetuximab did not reduce the number of papulopustular eruptions, and this was independent of the use of systemic tetracycline. No significant changes in the staining of EGFR or pEGFR were observed in the skin of the vitamin K3-treated area compared to the placebo area. CONCLUSION: The present data do not support any clinical or immunohistochemical benefit of using vitamin K3 cream for cetuximab-induced rash.

Acupuncture point injection treatment of primary dysmenorrhoea: a randomised, double blind, controlled study.

OBJECTIVE: To determine if injection of vitamin K3 in an acupuncture point is optimal for the treatment of primary dysmenorrhoea, when compared with 2 other injection treatments. SETTING: A Menstrual Disorder Centre at a public hospital in Shanghai, China. PARTICIPANTS: Chinese women aged 14-25 years with severe primary dysmenorrhoea for at least 6 months not relieved by any other treatment were recruited. Exclusion criteria were the use of oral contraceptives, intrauterine devices or anticoagulant drugs, pregnancy, history of abdominal surgery, participation in other therapies for pain and diagnosis of secondary dysmenorrhoea. Eighty patients with primary dysmenorrhoea, as defined on a 4-grade scale, completed the study. Two patients withdrew after randomisation. INTERVENTIONS: A double-blind, double-dummy, randomised controlled trial compared vitamin K3 acupuncture point injection to saline acupuncture point injection and vitamin K3 deep muscle injection. Patients in each group received 3 injections at a single treatment visit. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome was the difference in subjective perception of pain as measured by an 11 unit Numeric Rating Scale (NRS). Secondary measurements were Cox Pain Intensity and Duration scales and the consumption of analgesic tablets before and after treatment and during 6 following cycles. RESULTS: Patients in all 3 groups experienced pain relief from the injection treatments. Differences in NRS measured mean pain scores between the 2 active control groups were less than 1 unit (-0.71, CI -1.37 to -0.05) and not significant, but the differences in average scores between the treatment hypothesised to be optimal and both active control groups (1.11, CI 0.45 to 1.78) and (1.82, CI 1.45 to 2.49) were statistically significant in adjusted mixed-effects models. Menstrual distress and use of analgesics were diminished for 6 months post-treatment. CONCLUSIONS: Acupuncture point injection of vitamin K3 relieves menstrual pain rapidly and is a useful treatment in an urban outpatient clinic. TRIAL REGISTRATION NUMBER: NCT00104546; Results.

Design and rationale of a 16-week adjunctive randomized placebo-controlled trial of mitochondrial agents for the treatment of bipolar depression.

OBJECTIVE: Bipolar disorder places a significant burden on individuals, caregivers and family, and the broader community. Current treatments are believed to be more effective against manic symptoms, leaving a shortfall in recovery during the depressive phase of the illness. The current study draws on recent evidence suggesting that, in addition to increased oxidative load, alterations in mitochondrial function occur in bipolar disorder. METHODS: This 16-week study aims to explore the potential benefits of N-acetylcysteine (NAC) alone or in combination (CT) with selected nutraceuticals believed to enhance mitochondrial function. The study includes adults diagnosed with bipolar disorder currently experiencing an episode of depression. Participants are asked to take NAC, CT, or placebo in addition to any usual treatments. A post-discontinuation visit is conducted 4 weeks following the treatment phase. RESULTS: The primary outcome of the study will be mean change on the Montgomery-Asberg Depression Rating Scale. Secondary outcomes include functioning, substance use, mania ratings, and quality of life. Blood samples will be collected at baseline and week 16 to explore biochemical alterations following treatment. CONCLUSION: This study may provide a novel adjunctive treatment for bipolar depression. Analysis of biological samples may assist in understanding the therapeutic benefits and the underlying etiology of bipolar depression. TRIAL REGISTRATION: Australian and New Zealand Clinical Trial Registry ACTRN12612000830897.

Novel Vitamin K analogs suppress seizures in zebrafish and mouse models of epilepsy.

Epilepsy is a debilitating disease affecting 1-2% of the world's population. Despite this high prevalence, 30% of patients suffering from epilepsy are not successfully managed by current medication suggesting a critical need for new anti-epileptic drugs (AEDs). In an effort to discover new therapeutics for the management of epilepsy, we began our study by screening drugs that, like some currently used AEDs, inhibit histone deacetylases (HDACs) using a well-established larval zebrafish model. In this model, 7-day post fertilization (dpf) larvae are treated with the widely used seizure-inducing compound pentylenetetrazol (PTZ) which stimulates a rapid increase in swimming behavior previously determined to be a measurable manifestation of seizures. In our first screen, we tested a number of different HDAC inhibitors and found that one, 2-benzamido-1 4-naphthoquinone (NQN1), significantly decreased swim activity to levels equal to that of valproic acid, 2-n-propylpentanoic acid (VPA). We continued to screen structurally related compounds including Vitamin K3 (VK3) and a number of novel Vitamin K (VK) analogs. We found that VK3 was a robust inhibitor of the PTZ-induced swim activity, as were several of our novel compounds. Three of these compounds were subsequently tested on mouse seizure models at the National Institute of Neurological Disorders and Stroke (NINDS) Anticonvulsant Screening Program. Compound 2h reduced seizures particularly well in the minimal clonic seizure (6Hz) and corneal-kindled mouse models of epilepsy, with no observable toxicity. As VK3 affects mitochondrial function, we tested the effects of our compounds on mitochondrial respiration and ATP production in a mouse hippocampal cell line. We demonstrate that these compounds affect ATP metabolism and increase total cellular ATP. Our data indicate the potential utility of these and other VK analogs for the prevention of seizures and suggest the potential mechanism for this protection may lie in the ability of these compounds to affect energy production.

Alternative therapeutic approach to renal-cell carcinoma: induction of apoptosis with combination of vitamin K3 and D-fraction.

PURPOSE: Because of a dismal prognosis for advanced renal-cell carcinoma (RCC), an alternative therapeutic approach, using vitamin K3 (VK3) and D-fraction (DF) was investigated. VK3 is a synthetic VK derivative and DF is a bioactive mushroom extract, and they have been shown to have antitumor activity. We examined if the combination of VK3 and DF would exhibit the improved anticancer effect on RCC in vitro. MATERIALS AND METHODS: Human RCC, ACHN cell line, were treated with varying concentrations of VK3, DF, or a combination of the two. Cell viability was assessed at 72 hours by MTT assay. To explore the possible anticancer mechanism, studies on cell cycle, chromatin modifications, and apoptosis were conducted. RESULTS: VK3 alone led to a ~20% reduction in cell viability at 4 µM, while DF alone induced a 20% to 45% viability reduction at ≥ 500 µg/mL. A combination of VK3 (4 µM) and DF (300 µg/mL) led to a drastic >90% viability reduction, however. Cell cycle analysis indicated that VK3/DF treatment induced a G1 cell cycle arrest, accompanied by the up-regulation of p21(WAF1) and p27(Kip1). Histone deacetylase (HDAC) was also significantly (~60%) inactivated, indicating chromatin modifications. In addition, Western blot analysis revealed that the up-regulation of Bax and activation of poly-(ADP-ribose)-polymerase (PARP) were seen in VK3/DF-treated cells, indicating induction of apoptosis. CONCLUSIONS: The combination of VK3 and DF can lead to a profound reduction in ACHN cell viability, through a p21(WAF1)-mediated G1 cell cycle arrest, and ultimately induces apoptosis. Therefore, the combination of VK3/DF may have clinical implications as an alternative, improved therapeutic modality for advanced RCC.

The zebrafish as a model to study polycystic liver disease.

In the polycystic liver diseases (PLD), genetic defects initiate the formation of cysts in the liver and kidney. In rodent models of PLD (i.e., the PCK rat and Pkd2(WS25/-) mouse), we have studied hepatorenal cystic disease and therapeutic approaches. In this study, we employed zebrafish injected with morpholinos against genes involved in the PLD, including sec63, prkcsh, and pkd1a. We calculated the liver cystic area, and based on our rodent studies, we exposed the embryos to pasireotide [1 µM] or vitamin K3 [100 µM] and assessed the endoplasmic reticulum (ER) in cholangiocytes in embryos treated with 4-phenylbutyrate (4-PBA). Our results show that (a) morpholinos against sec63, prkcsh, and pkd1a eliminate expression of the respective proteins; (b) phenotypic body changes included curved tail and the formation of hepatic cysts in zebrafish larvae; (c) exposure of embryos to pasireotide inhibited hepatic cystogenesis in the zebrafish models; and (d) exposure of embryos to 4-PBA resulted in the ER in cholangiocytes resolving from a curved to a smooth appearance. Our results suggest that the zebrafish model of PLD may provide a means to screen drugs that could inhibit hepatic cystogenesis.