Active vitamin D derivatives (VDDs)—1α,25-dihydroxyvitamin D3/D2 and their synthetic analogs—are well-known inducers of cell maturation with the potential for differentiation therapy of acute myeloid leukemia (AML). However, their dose-limiting calcemic activity is a significant obstacle to using VDDs as an anticancer treatment. We have shown that different activators of the NF-E2-related factor-2/Antioxidant Response Element (Nrf2/ARE) signaling pathway, such as the phenolic antioxidant carnosic acid (CA) or the multiple sclerosis drug monomethyl fumarate (MMF), synergistically enhance the antileukemic effects of various VDDs applied at low concentrations in vitro and in vivo. This study aimed to investigate whether glutathione, the major cellular antioxidant and the product of the Nrf2/ARE pathway, can mediate the Nrf2-dependent differentiation-enhancing activity of CA and MMF in HL60 human AML cells. We report that glutathione depletion using L-buthionine sulfoximine attenuated the enhancing effects of both Nrf2 activators concomitant with downregulating vitamin D receptor (VDR) target genes and the activator protein-1 (AP-1) family protein c-Jun levels and phosphorylation. On the other hand, adding reduced glutathione ethyl ester to dominant negative Nrf2-expressing cells restored both the suppressed differentiation responses and the downregulated expression of VDR protein, VDR target genes, as well as c-Jun and P-c-Jun levels. Finally, using the transcription factor decoy strategy, we demonstrated that AP-1 is necessary for the enhancement by CA and MMF of 1α,25-dihydroxyvitamin D3-induced VDR and RXRα protein expression, transactivation of the vitamin D response element, and cell differentiation. Collectively, our findings suggest that glutathione mediates, at least in part, the potentiating effect of Nrf2 activators on VDDs-induced differentiation of AML cells, likely through the positive regulation of AP-1.

The inhalation of gasoline vapors (GV) is associated with developing various pathologies. Particularly, oil refinery and gas station workers are at a greater risk of developing lung cancer, kidney cancer, bladder cancer, and hematological disorders, including acute myeloid leukemia. Therefore, preventing the harmful effects of GV and alleviating their consequences appear to be important and timely issues. In this study, we investigated the potential of vitamin D3, turmeric powder, and their combination to ameliorate the toxicity of gasoline fumes in rats. Separate groups of animals fed with a standard rodent diet, with or without the supplementation of vitamin D3 (750 IU/kg body weight) and/or turmeric powder (0.5%, w/w, in food), were untreated or treated with GV (11.5 ± 1.3 cm3/h/m3/day) for 30, 60, or 90 days. Changes in the body weight were monitored weekly. Histological, biochemical, and hematological parameters were determined at the end of each treatment period. While the exposure of rats to GV resulted in a time-dependent reduction in body weight, supplementation with vitamin D3, but not with turmeric root powder or their combination, partially prevented weight loss. Macroscopical and histological analyses showed pronounced time-dependent changes in the organs and tissues of GV-treated rats. These included alveolar wall collapse in the lungs, the destruction of the lobular structure and hepatocytolysis in the liver, the shrinkage and fragmentation of glomeruli in the kidneys, and the disorganization of the lymphoid follicles in the spleen. However, co-treatment with the nutritional supplements tested, especially vitamin D3, noticeably alleviated the above conditions. This was accompanied by a significant improvement in the blood chemistry and hematological parameters. Collectively, our results demonstrate that the harmful effects of environmental exposure to GV can be reduced upon supplementation of vitamin D3. The fact that the protective activity of vitamin D3 alone was higher than that of turmeric root powder or the combined treatment suggests that combinations of these supplements may not always be more beneficial than each agent applied separately.

The physiological form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has a role in normal hematopoiesis, enhancing monocyte–macrophage differentiation. The focus in this chapter is on the antiproliferative, prodifferentiation, and cell survival effects of vitamin D derivatives (VDDs) studied in various myeloid leukemia cell lines and leukemic blasts ex vivo. The proposed mechanisms and signaling pathways by which VDDs induce cell type–specific and cell context–specific effects are described here. Clinical trials with 1,25(OH)2D3 have been performed for the treatment of preleukemia/myelodysplastic syndrome and acute myeloid leukemia (AML), but the doses effective in vitro cause hypercalcemia in vivo. However, numerous VDDs have been synthesized, which in model systems have reduced calcemic activity but an increased ability to induce cell differentiation and to inhibit proliferation of leukemic cells. The availability of new analogs combined with other differentiating or antiproliferative agents may demonstrate synergistic activity and offer improved therapy for AML.

Treatment of acute myeloid leukemia (AML) has not significantly improved in the past 40 years. The active form of vitamin D, 1.25-dihydroxyvitamin D3 (1,25D3), has strong in-vitro antileukemic effects at toxic concentrations in vivo. Several low-calcemic vitamin D analogs (VDAs) have been synthesized to reduce 1,25D3 toxicity. We have previously shown that plant polyphenolic antioxidants (PAOx) synergistically enhance the effects of 1,25D3 and VDAs at non-toxic doses. These effects were mediated b y the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activator protein-1 (AP-1) transcription factors. The study aimed to 1) Character-ize the combined effects of novel highly potent vascular disrupting agents with peroxisomal N(1) –acetyl-spermine/spermidine oxidase on differentiation and growth of acute myeloid leukemia cells; 2) Elucidate the role of the interplay between nuclear factor erythroid 2, activator protein-1, and vitamin D receptor (VDR) in the combined antileukemic effects of vascular disrupting agents and peroxisomal N (1)–acetyl-spermine/spermidine oxidase. This article presents the treatment of AML with pharmaceutical plants and vitamin D. Such techniques as culture dilution (HL60, U937) and Western Blotting were used. We determined the in-vitro antileukemic effects of novel plant vitamin D2-based VDAs with removed C-19 (19-nor analogs; PRI-5201 and PRI-5202) or with a side-chain modification at C-24 (24-cis analogs; PRI-1916 and PRI-1917) on HL60, U937, and MOLM-13 human AML cells. The differentiation potency of PRI-5201 and PRI-5202 was 1-2 orders of magnitude higher than that of 1,25D2 or 1,25D3, while the 24-cis modification was almost ineffective. The 19-nor VDA/CA combinations are promising for treating AML. In vivo, testing of these combinations is in progress. We suggest that direct or indirect Nrf2-mediated up regulation of AP-1 and VDR by PAOx increases the sensitivity of AML cells to low doses of 1,25D3 or VDAs.

The article presents the results of the study of vitamin D receptor (VDR) gene rs2228570 single nucleotide polymorphism (SNP) genotypes and individual alleles frequency among the Kazakh ethnic group representatives living in the Karaganda region. This SNP was determined by real-time polymerase chain reaction using TaqMan technology. The study relevance is due to the fact that genetic variations in rs2228570 affect the synthesis of the VDR protein and its activity as a transcription factor that regulates the expression of other genes. This mechanism determines the association of individual rs2228570 genotypes or alleles with susceptibility, course, and outcomes of various diseases. The polymorphism frequency may be depending on ethnicity. According to the study results, the most common genotypes of rs2228570 SNP among the Kazakhs were AG (32.8 %) and GG (25.2 %). The rarest are homozygotes TT (1.7 %) and CC (0.8 %). The frequency of all rs2228570 alleles was detected simultaneously in the present study for the first time. G became the predominant allele (51.3 %), less common was A (31.0 %), and the C and T alleles were the rarest (11.8 % and 5.9 %, respectively). The potential value of this SNP further study as a possible factor influencing the body’s susceptibility to various diseases, including COVID-19, is shown.

The purpose of our study was to evaluate the analgesic properties of continuous transversus abdominis plane (TAP) infusion with ropivacaine compared to placebo for postoperative analgesia in elective surgery of the abdominal aorta by retroperitoneal exposure.We conducted a prospective, single-center, randomized, double-blind study comparing a group of patients with a TAP catheter undoing ropivacaine infusion with a placebo group. Patients received a left retroperitoneal pararectal exposure for abdominal aortic surgery. A continuous infusion catheter was placed under visual control by the surgeon before closure and removed after 48 hr. All patients had postoperative patient-controlled analgesia with morphine. The primary endpoint was morphine consumption during the first 24 hr.The analysis included 25 patients in the placebo group and 24 in the ropivacaine group. The average morphine consumption during the first 24 hr was significantly different, with 31 ± 16 mg in the ropivacaine group and 41 ± 17 mg in the placebo group (P = 0.019). At 48 hr, morphine consumption was still lower in the ropivacaine group (42 ± 26 mg) than in the placebo group (64 ± 25 mg) (P = 0.003). The opioid narcotic-related side effects of opioid infusion (postoperative nausea and vomiting, constipation) and length of hospital stay were similar in both populations.Our study showed that continuous TAP block with ropivacaine via surgically inserted catheter significantly decreased morphine consumption at 24 and 48 hr after elective abdominal aortic surgery by retroperitoneal exposure.

Acute myeloid leukemia (AML) is an aggressive type of leukemia, characterized by the accumulation of highly proliferative blasts with a disrupted myeloid differentiation program. Current treatments are ineffective for most patients, partly due to the genetic heterogeneity of AML. This is driven by genetically distinct leukemia stem cells, resulting in relapse even after most of the tumor cells are destroyed. Thus, personalized treatment approaches addressing cellular heterogeneity are urgently required. Reconstruction of Transcriptional regulatory Networks (RTN) is a tool for inferring transcriptional activity in patients with various diseases. In this study, we applied this method to transcriptome profiles of AML patients to test if it provided additional information for the interpretation of transcriptome data. We showed that when RTN results were added to RNA-seq results, superior clusters were formed, which were more homogenous and allowed the better separation of patients with low and high survival rates. We concluded that the external knowledge used for RTN analysis improved the ability of unsupervised machine learning to find meaningful patterns in the data.

Novel derivatives of aminophenyl-1,4-naphthoquinones, in which a pyrrolidine group was added to the naphthoquinone ring, were synthesized and investigated for the mechanisms of leukemic cell killing. The novel compounds, TW-85 and TW-96, differ in the functional (methyl or hydroxyl) group at the para-position of the aminophenyl moiety. TW-85 and TW-96 were found to induce concentration- and time-dependent apoptotic and/or necrotic cell death in human U937 promonocytic leukemia cells but only TW-96 could also kill K562 chronic myeloid leukemia cells and CCRF-CEM lymphoblastic leukemia cells. Normal peripheral blood mononuclear cells were noticeably less responsive to both compounds than leukemia cells. At low micromolar concentrations used, TW-85 killed U937 cells mainly by inducing apoptosis. TW-96 was a weaker apoptotic agent in U937 cells but proved to be cytotoxic and a stronger inducer of necrosis in all three leukemic cell lines tested. Both compounds induced mitochondrial permeability transition pore opening, cytochrome c release, and caspase activation in U937 cells. Cytotoxicity induced by TW-96, but not by TW-85, was associated with the elevation of the cytosolic levels of reactive oxygen species (ROS). The latter was attenuated by diphenyleneiodonium, indicating that NADPH oxidase was likely to be the source of ROS generation. Activation of p38 MAPK by the two agents appeared to prevent necrosis but differentially affected apoptotic cell death in U937 cells. These results further expand our understanding of the structure–activity relationship of aminophenyl-1,4-naphthoquinones as potential anti-leukemic agents with distinct modes of action.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by uncontrolled proliferation of immature myeloid progenitors. Here, we report the in vitro antileukemic effects of the sesquiterpene thioalkaloid-enriched fraction of the Nuphar lutea leaf extract (NUP) and a purified thioalkaloid 6,6′-dihydroxythiobinupharidine (DTBN). Treatment with 0.3–10 µg/mL NUP caused a dose- and time-dependent reduction in proliferation and viability of human AML cells (KG-1a, HL60 and U937). This was associated with apoptosis induction manifested by annexin-V/propidium iodide binding as well as cleavage of caspases 8, 9, and 3 as well as poly (ADP-ribose) polymerase. Caspase-dependence of the apoptotic effect was confirmed using the pan-caspase inhibitor Q-VD-OPH. NUP induced significant biphasic changes in the cytosolic levels of reactive oxygen species (ROS) compared to untreated cells—a decrease at early time points (2–4 h) and an increase after a longer incubation (24 h). ROS accumulation was accompanied by lowering the cellular glutathione (GSH) levels. In addition, NUP treatment resulted in elevation of the cytosolic Ca2+ (Ca2+cyt) levels. The thiol antioxidant and glutathione precursor N-acetyl cysteine prevented NUP-induced ROS accumulation and markedly inhibited apoptosis. A similar antiapoptotic effect was obtained by Ca2+cyt chelating using BAPTA. These data indicate that NUP-induced cell death is mediated, at least in part, by the induction of oxidative stress and Ca2+cyt accumulation. However, a substantial apoptotic activity of pure DTBN (0.05–0.25 µg/mL), was found to be independent of cytosolic ROS or Ca2+, suggesting that alternative mechanisms are involved in DTBN-induced cytotoxicity. Notably, neither NUP nor DTBN treatment significantly induced cell death of normal human peripheral blood mononuclear cells. Our results provide the basis for further investigation of the antileukemic potential of NUP and its active constituents.

Host genes act as a factor related to susceptibility and resistance to viral infections. The article provides a description of modern scientific studies devoted to the study of the role of the ACE2, TMPRSS2 genes, and their single-nucleotide polymorphisms in infection with the SARS-CoV-2 virus. SNPs of the ACE2 gene, TMPRSS2 can affect the penetration of SARS-CoV-2 into the cell. In addition, the study of these polymorphisms will determine the predisposition of an individual to the disease COVID–19, or the nature of its course. Based on the literature sources, the role of angiotensin-converting enzyme 2 and transmembrane proteases in the participation of the SARS-CoV-2 virus penetration process with the body cells is noted. Other functions that ACE2 and TMPRSS2 receptors perform in the human body are also described. The characteristics of two genes and their fairly well-known polymorphisms are given. The tissues and organs in which genes are expressed are marked. Information on the frequency of alleles of genetic variants of genes in different populations is shown. In addition to describing the relationship of gene polymorphisms with the disease caused by SARSCoV-2, information is provided on the association of these genetic variations with diseases of the blood vascular system and oncological diseases.

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality.

The article presents the characteristics of the main vitamin D receptor (VDR) gene polymorphisms: rs2228570 (FokI), rs731236 (TaqI), rs1544410 (BsmI) and rs7975232 (ApaI). The role of the vitamin D hormonally active form (1,25(OH)2D3, calcitriol) as a transcription factor regulating gene expression in target cells by binding to the vitamin D receptor protein is described. The immunomodulatory and mediating effect of VDRs on the biological functions of the human body has been noted. A description of the vitamin D receptor gene and its polymorphic character have been provided. The analysis of the four most significant single nucleotide polymorphisms (SNPs) of the VDR gene was carried out. A detailed description of each polymorphism, its genomic position, the nature of interaction with other polymorphisms of the vitamin D receptor gene, as well as its effect on the structure and activity of the VDR protein were given. The analysis of the indicated single-nucleotide polymorphisms allelic composition was conducted according to the literature and specialized SNP databases. The frequency of each polymorphism individual alleles occurrence, as well as their influence on the predisposition and course of various diseases, were studied. The need for further studies of VDR gene polymorphisms, their allelic composition and prevalence was designated. It is also necessary to study the possibilities of their potential use as genetic markers for such relevant but little-studied pathologies as COVID-19.

Plant phenolic compounds have shown the ability to cooperate with one another at low doses in producing enhanced anticancer effects. This may overcome the limitations (e.g., poor bioavailability and high-dose toxicity) in developing these agents as cancer medicines. We have previously reported that the hydroxycinnamic acid derivative (HCAD) methyl-4-hydroxycinnamate and the phenolic diterpene carnosic acid (CA) can synergistically induce massive calcium-dependent apoptosis in acute myeloid leukemia (AML) at non-cytotoxic concentrations of each agent. Here, we explored the chemical nature of the synergy between HCADs and either CA, in inducing cytotoxicity, or the active metabolite of vitamin D (calcitriol), in enhancing the differentiation of AML cells. This was done by determining the structure–activity relationship of a series of hydroxycinnamic acids and their derivatives (methyl hydroxycinnamates and hydroxybenzylideneacetones) in combination with CA or calcitriol. The HCAD/CA synergy required the following critical structural elements of an HCAD molecule: (a) the para-hydroxyl on the phenolic ring, (b) the carbon C7–C8 double bond, and (c) the methyl-esterified carboxyl. Thus, the only HCADs capable of synergizing with CA were found to be methyl-4-hydroxycinnamate and methyl ferulate, which also most potently enhanced calcitriol-induced cell differentiation. Notably, the C7–C8 double bond was the major requirement for this HCAD/calcitriol cooperation. Our findings may contribute to the rational design of novel synergistically acting AML drugs based on prototype combinations of HCADs with other agents studied here.

The authors consider the basic concepts of HPV-induced carcinogenesis and the molecular differences found among types of HPV and intra-type variants, and give their clinical and functional consequences. Human papillomavirus (HPV) is a diverse group of small DNA viruses, some of which have been extensively studied over the past three decades due to their carcinogenic potential. The persistence of viral infections and the uncontrolled expression of the E6 and E7 viral oncogenes are critical events in the transformation process. It is important to note that viral types are specific for each type of cell and usually cause various types of lesions, benign or malignant. Recognizing the critical role that certain specific types of HPV play in the development of cervical cancer is very important for their prevention and public health strategies for cervical cancer, which are still the leading cause of death among cancer patients in many countries.

Hepatocellular carcinoma (HCC) has increasing worldwide incidence but when unresectable lacks curative options. Treatment with a kinase inhibitor Sorafenib (Sf), while initially effective, results in only short increases in patient survival, thus there is a need for improved treatment regimens. Numerous treatment regimens have been explored wherein Sf is combined with other agents, such as non-toxic botanicals like Curcumin or Silibinin. Recently, we have shown that carnosic acid (CA), a component of the food preservative Rosemary Extract, can markedly enhance the cytotoxic actions of Sf in several cell lines derived from HCC, but not in the cell line Hu1545 derived from normal hepatocytes. CA has been shown to enhance Sf-induced cell death in the neoplastic cell lines, principally due to the composite of increased apoptosis and cytotoxic autophagy. In the present study we focused on the mechanisms that underlie the reduced proliferation and survival of HCC cells when CA is added to Sf and how this relates to the increase in Sf-induced DNA damage as well as to the elevation of cytoplasmic levels of reactive oxygen species (ROS). Importantly, the elevation of ROS levels induced by Sf was increased by adding CA. We found that CA enhanced Sf-induced prolongation of cell cycle, and the overall decrease in cell growth was associated with reduced activation of both STAT3 transcription factor (TF) and extracellular signal-regulated protein kinase (Erk)1/2. Our data suggest that a regimen incorporating CA, an inexpensive and non-toxic food additive, in the treatment of advanced HCC merits clinical evaluation.

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality worldwide. Its incidence is increasing, and currently there is no curative therapy for it in advanced cases. Sorafenib (Sf), a multikinase inhibitor, is an FDA approved drug for the treatment of unresectable hepatomas, but the improvement in patient survival is very short, so new approaches are needed to supplement the existing regimens. In this study we show that the addition of a plant antioxidant Carnosic acid (CA) and a vitamin D2 derivative Doxercalciferol (D2) to Sf can markedly enhance its cytotoxic effect on HCC cell lines Huh7 (Sf-sensitive) and HCO2 (partially Sf-resistant). In contrast, similar combinatorial treatment of Hu1545 cells (immortalized human hepatocytes) results in only a minimal increase in cell death (CD). The combination Sf-CA/D2 increases HCC cell death by enhancing both apoptosis and cytotoxic autophagy (AuPh). Apoptosis was shown by Annexin V binding analysis and by the increased levels of apoptosis-related proteins BIM, cleaved caspases 9 and 3. AuPh was demonstrated by increased cytoplasmic vacuolation and elevated protein levels of Beclin1, Atg3, and LC3-II. Individually, both CA and D2 enhance Sf-induced CD, but CA produces the predominant effect. These effects were significantly reduced by the addition of the Caspase 3 inhibitor Z-DEVD-fmk or the AuPh inhibitor Chloroquine, and by Beclin1 depletion. A pilot experiment with mice bearing a Huh7 HCC cell xenograft showed markedly increased survival of the animals when CA was added to Sf. These results suggest that CA alone or with D2 may increase the survival of Sf-treated HCC patients. Supported by NIH grants 1R01DK110024 (to CL) and 5R01 44722-27 (to GPS). Citation Format: Xuening Wang, Qunfeng Wu, Kien Pham, Aesis Luna, Michael Danilenko, Chen Liu, George P. Studzinski. Sorafenib-mediated apoptotic and autophagic cell death is increased by carnosic acid and a vitamin D2 analog in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1811.

Low blood levels of the vitamin D metabolite 25-hydroxyvitamin D [25(OH)D] have been associated with an increased risk and poorer outcomes of various cancers, including hematological malignancies. The Central Kazakhstan area has a relatively high incidence rate of leukemia. However, the relationship between vitamin D status and leukemia or other types of cancer in Kazakhstan has not yet been addressed. Therefore, in this first pilot single-center study conducted in Central Kazakhstan, we compared plasma levels of 25(OH)D and the vitamin D receptor (VDR) gene expression levels in peripheral blood mononuclear cells of patients with leukemia and demographically matching healthy volunteers. The levels of 25(OH)D in patients were found to be significantly lower (10.8 ± 7.0 ng/mL; n = 31) than in healthy subjects (21.6 ± 7.8 ng/mL; n = 34; p < 0.0001). A similar difference was observed in both younger (<60 years old) and older (>60 years old) participants, though there was no association between 25(OH)D concentration and age within the patient group. In female patients, 25(OH)D levels were significantly lower than in male patients (p = 0.04). No significant seasonal variations of 25(OH)D were observed in either the patient or the control group. VDR gene expression levels appeared to be similar in leukemia patients and healthy subjects, and no correlation between the cellular VDR expression and plasma 25(OH)D concentrations was observed in either group of participants. We did not observe a significant association of 25(OH)D or VDR levels and overall survival of leukemia patients. This observational study conducted for the first time in Kazakhstan supports previous findings demonstrating reduced blood 25(OH)D levels in cancer (leukemia) patients. Larger studies are required to determine whether low 25(OH)D plasma concentrations represent a risk factor for leukemia development and/or progression.

Acute myeloid leukemia (AML) is one of the deadliest hematological malignancies without effective treatment for most patients. Vitamin D derivatives (VDDs) - active metabolites 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) and their analogs - are differentiation-inducing agents which have potential for the therapy of AML. However, calcemic toxicity of VDDs limits their clinical use at doses effective against cancer cells in vivo. Here, we demonstrate that in AML cell cultures, moderate pro-differentiation effects of low concentrations of VDDs can be synergistically enhanced by structurally distinct compounds known to activate the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (NFE2L2 or Nrf2). Particularly, dimethyl fumarate (DMF), which is clinically approved for the treatment of multiple sclerosis and psoriasis, strongly cooperated with 1,25D3, PRI-5100 (19-nor-1,25D2; paricalcitol) and PRI-5202 (a double-point modified 19-nor analog of 1,25D2). The pro-differentiation synergy between VDDs (1,25D3 or PRI-5202) and Nrf2 activators (DMF, tert-butylhydroquinone or carnosic acid) was associated with a cooperative upregulation of the protein levels of the vitamin D receptor (VDR) and Nrf2 as well as increased mRNA expression of their respective target genes. These data support the notion that VDDs and Nrf2 activators synergize in inducing myeloid cell differentiation through the cooperative activation of the VDR and Nrf2/antioxidant response element signaling pathways. We have previously reported that PRI-5202 is more potent by approximately two orders of magnitude than 1,25D3 as a differentiation inducer in AML cell lines. In this study, we found that PRI-5202 was also at least 5-fold less calcemic in healthy mice compared to both its direct precursor PRI-1907 and 1,25D3. In addition, PRI-5202 was remarkably more resistant against degradation by the human 25-hydroxyvitamin D3-24-hydroxylase than both 1,25D2 and 1,25D3. Importantly, using a xenograft mouse model we demonstrated that co-administration of PRI-5202 and DMF resulted in a marked cooperative inhibition of human AML tumor growth without inducing treatment toxicity. Collectively, our findings provide a rationale for clinical testing of low-toxic VDD/DMF combinations as a novel approach for differentiation therapy of AML.

Acute myeloid leukemia (AML) is a malignant hematopoietic disease with poor prognosis for most patients. Conventional chemotherapy has been the standard treatment approach for AML in the past 40 years with limited success. Although, several targeted drugs were recently approved, their long-term impact on survival of patients with AML is yet to be determined. Thus, it is still necessary to develop alternative therapeutic approaches for this disease. We have previously shown a marked synergistic anti-leukemic effect of two polyphenols, curcumin (CUR) and carnosic acid (CA), on AML cells in-vitro and in-vivo. In this study, we identified another phenolic compound, methyl 4-hydroxycinnamate (MHC), which among several tested phytochemicals could uniquely cooperate with CA in killing AML cells, but not normal peripheral blood mononuclear cells. Notably, our data revealed striking phenotypical and mechanistic similarities in the apoptotic effects of MHC+CA and CUR+CA on AML cells. Yet, we show that MHC is a non-fluorescent molecule, which is an important technical advantage over CUR that can interfere in various fluorescence-based assays. Collectively, we demonstrated for the first time the antileukemic activity of MHC in combination with another phenolic compound. This type of synergistically acting combinations may represent prototypes for novel antileukemic therapy.

Acute myeloid leukemia (AML) has a poor prognosis and requires new approaches for treatment. We have reported that a combination of vitamin D‐based cell differentiation agents (doxercalciferol/carnosic acid [D2/CA]) added following the cytotoxic drug arabinocytosine (AraC) increases AML cell death (CD), a model for improved therapy of this disease. Because AraC‐induced CD is known to involve reactive oxygen species (ROS) generation, here we investigated if the modulation of cellular REDOX status plays a role in the enhancement of cell death (ECD) by D2/CA. Using thiol antioxidants, such as N‐acetyl cysteine (NAC), we found a significant inhibition of ECD, yet this occurred in the absence of any detectable change in cellular ROS levels. In contrast, NAC reduced the vitamin D receptor (VDR) abundance and its signaling of ECD. Importantly, VDR knockdown and NAC similarly inhibited ECD without producing an additive effect. Thus, the proposed post‐AraC therapy may be compromised by agents that reduce VDR levels in AML blasts.