PBK/TOPK Is a Favorable Prognostic Biomarker Correlated with Antitumor Immunity in Colon Cancers.

Immune checkpoint inhibitor therapy has proven efficacy in a subset of colon cancer patients featuring a deficient DNA mismatch repair system or a high microsatellite instability profile. However, there is high demand for more effective biomarkers to expand the colon cancer population responding to ICI therapy. PBK/TOPK, a serine/threonine kinase, plays a role in cell cycle regulation and mitotic progression. Here, we investigated the correlation between PBK/TOPK expression and tumor immunity and its prognostic value in colon cancer. Based on large-scale bioinformatics analysis, we discovered that elevated PBK/TOPK expression predicted a favorable outcome in patients with colon cancer and was positively associated with immune infiltration levels of CD8+ T cells, CD4+ T cells, natural killer cells, and M1 macrophages. In contrast, a negative correlation was found between PBK/TOPK expression and immune suppressor cells, including regulatory T cells and M2 macrophages. Furthermore, the expression of PBK/TOPK was correlated with the expression of T-cell cytotoxicity genes in colon cancer. Additionally, high PBK/TOPK expression was associated with mutations in DNA damage repair genes, and thus with increased tumor mutation and neoantigen burden. These findings suggest that PBK/TOPK may serve as a prognostic and predictive biomarker for immunotherapy in colon cancer.

The sulfiredoxin-peroxiredoxin redox system regulates the stemness and survival of colon cancer stem cells.

Cancer stem cells (CSCs) initiate tumor formation and are known to be resistant to chemotherapy. A metabolic alteration in CSCs plays a critical role in stemness and survival. However, the association between mitochondrial energy metabolism and the redox system remains undefined in colon CSCs. In this study, we assessed the role of the Sulfiredoxin-Peroxiredoxin (Srx-Prx) redox system and mitochondrial oxidative phosphorylation (OXPHOS) in maintaining the stemness and survival of colon CSCs. Notably, Srx contributed to the stability of PrxI, PrxII, and PrxIII proteins in colon CSCs. Increased Srx expression promoted the stemness and survival of CSCs and was important for the maintenance of the mitochondrial OXPHOS system. Furthermore, Nrf2 and FoxM1 led to OXPHOS activation and upregulated expression of Srx-Prx redox system-related genes. Therefore, the Nrf2/FoxM1-induced Srx-Prx redox system is a potential therapeutic target for eliminating CSCs in colon cancer.

Inhibition of melanogenesis by Aster yomena callus pellet extract in melanoma cells and patients with skin pigmentation.

Plant tissue culture holds immense potential for the production of secondary metabolites with various physiological functions. We recently established a plant tissue culture system capable of producing secondary metabolites from Aster yomena. This study aimed to uncover the mechanisms underlying the potential therapeutic effects of Aster yomena callus pellet extract (AYC-P-E) on photoaging-induced skin pigmentation. Excessive melanogenesis was induced in B16F10 melanoma cells using α-melanocyte stimulating hormone (α-MSH). The effects of AYC-P-E treatment on melanin biosynthesis inducers and melanin synthesis inhibition were assessed. Based on the results, a clinical study was conducted in subjects with skin pigmentation. AYC-P-E inhibited melanogenesis in α-MSH-treated B16F10 cells, accompanied by decreased mRNA and protein expression of melanin biosynthesis inducers, including cyclic AMP response element-binding protein (CREB), tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), and TRP-2. This anti-melanogenic effect was mediated by mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) phosphorylation. Treatment of subjects with skin pigmentation with AYC-P-E-containing cream formulations resulted in 3.33%, 7.06%, and 8.68% improvement in the melanin levels at 2, 4, and 8 weeks, respectively. Our findings suggest that AYC-P-E inhibits excessive melanogenesis by activating MEK/ERK and AKT signaling, potentiating its cosmetic applications in hyperpigmentation treatment.

Overexpression of VlPRX21 and VlPRX35 genes in Arabidopsis plants leads to bioconversion of trans-resveratrol to δ-viniferin.

Stilbenes, including resveratrol and viniferins, a small family of polyphenols, are considered the most important phytoalexin group in Vitis species. In a previous study, we found that co-treatment of methyl jasmonate (MJ) and stevioside (STE) resulted in enhanced extracellular production of viniferins in grapevine cell suspension cultures. Thus, to further understand the mechanisms of viniferin production in grapevine cell cultures, we performed transcriptome analysis and isolated seven candidates of grapevine peroxidase genes (VlAPX6, VlGPX5, VlPRX13, VlPRX21, VlPRX35, VlPRX40, and VlPRX50). Bioconversion of trans-resveratrol to δ-viniferin was examined using crude protein extracts isolated from agroinfiltration-based transient expression of VlPRXs in Nicotiana benthamiana. In addition, we found that crude protein extracts from VlPRX21-, VlPRX35-, and VlPRX40-overexpressing (OX) transgenic Arabidopsis plants led to the conversion of trans-resveratrol to δ-viniferin. We found that in vitro experiments with crude protein extracts from VlPRX21-OX and VlPRX35-OX Arabidopsis plants catalyzed the dimerization of trans-resveratrol to δ-viniferin. Our results suggest that VlPRX21 and VlPRX35 encode functional grapevine class III peroxidases and catalyze the oxidative dimerization of trans-resveratrol to form δ-viniferin in grapevine.

Pharmacological inhibition of androgen receptor expression induces cell death in prostate cancer cells.

The androgen receptor (AR) plays an important role in the pathogenesis and development of prostate cancer (PCa). Mostly, PCa progresses to androgen-independent PCa, which has activated AR signaling from androgen-dependent PCa. Thus, inhibition of AR signaling may be an important therapeutic target in androgen-dependent and castration-resistant PCa. In this study, we determined the anticancer effect of a newly found natural compound, sakurasosaponin (S-saponin), using androgen-dependent and castration-resistant PCa cell lines. S-saponin induces mitochondrial-mediated cell death in both androgen-dependent (LNCaP) and castration-resistant (22Rv1 and C4-2) PCa cells, via AR expression. S-saponin treatment induces a decrease in AR expression in a time- and dose-dependent manner and a potent decrease in the expression of its target genes, including prostate-specific antigen (PSA), transmembrane protease, serin 2 (TMPRSS2), and NK3 homeobox 1 (NKX3.1). Furthermore, S-saponin treatment decreases B-cell lymphoma-extra large (Bcl-xL) and mitochondrial membrane potential, thereby increasing the release of cytochrome c into the cytosol. Moreover, Bcl-xL inhibition and subsequent mitochondria-mediated cell death caused by S-saponin were reversed by Bcl-xL or AR overexpression. Interestingly, S-saponin-mediated cell death was significantly reduced by a reactive oxygen species (ROS) scavenger, N-acetylcystein. Animal xenograft experiments showed that S-saponin treatment significantly reduced tumor growth of AR-positive 22Rv1 xenografts but not AR-negative PC-3 xenografts. Taken together, for the first time, our results revealed that S-saponin induces mitochondrial-mediated cell death in androgen-dependent and castration-resistant cells through regulation of AR mechanisms, including downregulation of Bcl-xL expression and induction of ROS stress by decreasing mitochondrial membrane potential.

Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells.

Malignant melanoma is the most life-threatening neoplasm of the skin. Despite the increase in incidence, melanoma is becoming more resistant to current therapeutic agents. The bioactive compound frugoside has been recently reported to inhibit growth when used in various cancer cells. However, this effect has not been demonstrated in melanoma. Here, we found that frugoside inhibited the rate of reduction of hyperoxidized peroxiredoxins (Prxs) by downregulating sulfiredoxin (Srx) expression. Furthermore, frugoside increased the accumulation of sulfinic Prxs and reactive oxygen species (ROS) and stimulated p-p38 activation, resulting in the mitochondria-mediated death of M14 and A375 human melanoma cells. The mitochondria-mediated cell death induced by frugoside was inhibited by the overexpression of Srx and antioxidants, such as N-acetyl cysteine and diphenyleneiodonium. In addition, we observed that frugoside inhibited tumor growth without toxicity through a M14 xenograft animal model. Taken together, our findings reveal that frugoside exhibits a novel antitumor effect based on a ROS-mediated cell death in melanoma cells, which may have therapeutic implications.

Modulation of Mitochondrial ERß Expression Inhibits Triple-Negative Breast Cancer Tumor Progression by Activating Mitochondrial Function.

BACKGROUND/AIMS: Breast cancer is a clinically and molecularly heterogeneous disease. Patients with triple-negative breast cancer (TNBC) have poorer outcomes than those with other breast cancer subtypes due to lack of effective molecular targets for therapy. The present study aimed to the identification of estrogen receptor (ER)ß as a novel mitochondrial target in TNBC cells, together with underlying mechanisms. METHODS: Expression of ERß in clinical breast samples were examined by qRT-PCR, immunohistochemistry and immunoblotting. Subcellular distribution and binding of ERß-Grp75 was determined by confocal microscopic analysis, co-immunoprecipitation experiments, and limited-detergent extraction of subcellular organelles. The effect of mitocondrial ERß(mitoERß) overexpression on cell proliferation and cell cycle distribution were assessed CCK-8 assays and FACS. Mitochondrial ROS, membrane potential, and Ca²âº level were measured using the specific fluorescent probes Mito-Sox, TMRE, and Rhod-2AM. The tumorigenic effect of mitoERß overexpression was assessed using an anchorage-independent growth assay, sphere formation and a mouse orthotopic xenograft model. RESULTS: ERß expression was lower in tumor tissue than in adjacent normal tissue of patients with breast cancer, and low levels of mitochondrial ERß (mitoERß) also were associated with increased tumor recurrence after surgery. Overexpression of mitoERß inhibited the proliferation of TNBC cells and tumor masses in an animal model. Moreover, overexpression of mitoERß increased ATP production in TNBC cells and normal breast MCF10A cells, with the latter completely reversed by mitoERß knockdown in MCF10A cells. Grp75 was found to positively regulate ERß translocation into mitochondria via a direct interaction. Coimmunoprecipitation and subcellular fractionation experiments revealed that ERß-Grp75 complex is stable in mitochondria. CONCLUSION: These results suggest that the up-regulation of mitoERß in TNBC cells ensures proper mitochondrial transcription, activating the OXPHOS system to produce ATP. Studying the effects of mitoERß on mitochondrial activity and specific mitochondrial gene expression in breast cancer might help predict tumor recurrence, inform clinical decision-making, and identify novel drug targets in the treatment of TNBC.

Pharmacologic inhibition of AKT leads to cell death in relapsed multiple myeloma.

Multiple myeloma (MM) is a neoplastic plasma cell disorder with high disease recurrence rates. Novel therapeutic approaches capable of improving outcomes in patients with MM are urgently required. The AKT signalling plays a critical regulatory role in MM pathophysiology, including survival, proliferation, metabolism, and has emerged as a key therapeutic target. Here, we identified a novel AKT inhibitor, HS1793, and defined its mechanism of action and clinical significance in MM. HS1793 disrupted the interaction between AKT and heat shock protein 90, resulting in protein phosphatase 2A-modulated phosphorylated-AKT (p-AKT) reduction. Moreover, we observed reductions in the kinase activity of the AKT downstream target, IκB kinase alpha, and the transcriptional activity of nuclear factor kappa B, which induced mitochondria-mediated cell death in MM cells exclusively. We confirmed the cytotoxicity and specificity of HS1793 via PET-CT imaging of a metastatic mouse model generated using human MM cells. We also evaluated the cytotoxic effects of HS1793 in primary and relapsed MM cells isolated from patients. Thus, HS1793 offers great promise in eliminating MM cells and improving therapeutic responses in primary and relapsed/refractory MM patients.

Peroxiredoxin 3 maintains the survival of endometrial cancer stem cells by regulating oxidative stress.

Cancer stem cell (CSC)-targeted therapy could reduce tumor growth, recurrence, and metastasis in endometrial cancer (EC). The mitochondria of CSCs have been recently found to be an important target for cancer treatment, but the mitochondrial features of CSCs and their regulators, which maintain mitochondrial function, remain unclear. Here, we investigated the mitochondrial properties of CSCs, and identified specific targets for eliminating CSCs in EC. We found that endometrial CSCs displayed higher mitochondrial membrane potential, Ca2+, reactive oxygen species, ATP levels, and oxygen consumption rates than non-CSCs. Further, we also verified that mitochondrial peroxiredoxin 3 (Prx3) was upregulated, and that it contributed to the survival of CSCs in EC. The knockdown of the Prx3 gene resulted not only in decreased sphere formation, but also reduced the viability of endometrial CSCs, by causing mitochondrial dysfunction. Furthermore, we found that the forkhead box protein M1 (FoxM1), an important transcriptional factor, is overexpressed in patients with EC. FoxM1 expression correlates with elevated Prx3 expression levels, in agreement with the tumorigenic ability of Prx3 in endometrial CSCs. Taken together, our findings indicate that human endometrial CSCs have enhanced mitochondrial function compared to that of endometrial tumor cells. Endometrial CSCs show increased expression of the mitochondrial Prx3, which is required for the maintenance of mitochondrial function and survival, and is induced by FoxM1. Based on our findings, we believe that these proteins might represent valuable therapeutic targets and could provide new insights into the development of new therapeutic strategies for patients with endometrial cancer.

Glucocorticoid receptor positively regulates transcription of FNDC5 in the liver.

Irisin is secreted by skeletal muscle during exercise and influences energy and metabolic homeostasis. This hormone is a cleaved and secreted fragment of fibronectin type III domain-containing 5 (FNDC5). Elucidation of the FNDC5 gene regulation mechanism is necessary to clarify the function of irisin as a potential therapeutic target in human metabolic diseases. Thus, we investigated the genetic and epigenetic mechanisms that regulate expression of the FNDC5 gene. FNDC5 mRNA was strong expressed in major energy-dependent human tissues, including heart, brain, liver, and skeletal muscle. Promoter analysis of the FNDC5 gene revealed that the core promoter region of the FNDC5 gene contained one CpG island that was located just upstream of the transcriptional start site for variants 2 and 3. Treatment with the histone deacetylase inhibitor sodium butyrate and the demethylating agent 5-azacytidine increased mRNA expression of FNDC5 in Huh7 cells. Prediction of transcription factor binding sites suggested that the glucocorticoid receptor was involved in the regulation of FNDC5 expression, and indeed, cortisol treatment increased mRNA expression of FNDC5 in Huh7 cells. Collectively, these findings offer insight into the genetic and epigenetic regulation of FNDC5, providing the initial steps required for understanding the role of irisin in the metabolic homeostasis.

Enhanced production of resveratrol derivatives in tobacco plants by improving the metabolic flux of intermediates in the phenylpropanoid pathway.

The biosynthesis of flavonoids such as anthocyanin and stilbenes has attracted increasing attention because of their potential health benefits. Anthocyanins and stilbenes share common phenylpropanoid precursor pathways. We previously reported that the overexpression of sweetpotato IbMYB1a induced anthocyanin pigmentation in transgenic tobacco (Nicotiana tabacum) plants. In the present study, transgenic tobacco (Nicotiana tabacum SR1) plants (STS-OX and ROST-OX) expressing the RpSTS gene encoding stilbene synthase from rhubarb (Rheum palmatum L. cv. Jangyeop) and the RpSTS and VrROMT genes encoding resveratrol O-methyltransferase from frost grape (Vitis riparia) were generated under the control of 35S promoter. Phenotypic alterations in floral organs, such as a reduction in floral pigments and male sterility, were observed in STS-OX transgenic tobacco plants. However, we failed to obtain STS-OX and ROST-OX plants with high levels of resveratrol compounds. Therefore, to improve the production of resveratrol derivatives in plants, we cross-pollinated flowers of STS-OX or ROST-OX and IbMYB1a-OX transgenic lines (SM and RSM). Phenotypic changes in vegetative and reproductive development of SM and RSM plants were observed. Furthermore, by HPLC and LC-MS analyses, we found enhanced production of resveratrol derivatives such as piceid, piceid methyl ether, resveratrol methyl ether O-hexoside, and 5-methyl resveratrol-3,4'-O-ß-D-diglucopyranoside in SM and RSM cross-pollinated lines. Here, total contents of trans- and cis-piceids ranged from approximately 104-240 µg/g fresh weight in SM (F2). Collectively, we suggest that coexpression of RpSTS and IbMYB1a via cross-pollination can induce enhanced production of resveratrol compounds in plants by increasing metabolic flux into stilbenoid biosynthesis.

Mitochondrial metabolism in cancer stem cells: a therapeutic target for colon cancer.

It has been proposed that the selective elimination of cancer stem cells (CSCs) using targeted therapy could greatly reduce tumor growth, recurrence, and metastasis. To develop effective therapeutic targets for CSC elimination, we aimed to define the properties of CSC mitochondria, and identify CSC-mitochondria- specific targets in colon cancer. We found that colon CSCs utilize mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP. We also found that forkhead box protein 1 (FOXM1)-induced peroxiredoxin 3 (PRDX3) maintains the mitochondrial function, and the FOXM1/PRDX3 mitochondrial pathway maintains survival of colon CSCs. Furthermore, FOXM1 induces CD133 (PROM1/prominin 1) expression, which maintains the stemness of colon CSCs. Together, our findings indicate that FOXM1, PRDX3, and CD133 are potential therapeutic targets for the elimination of CSCs in colon cancer.

FOXM1-Induced PRX3 Regulates Stemness and Survival of Colon Cancer Cells via Maintenance of Mitochondrial Function.

BACKGROUND & AIMS: Reagents designed to target cancer stem cells (CSCs) could reduce tumor growth, recurrence, and metastasis. We investigated the mitochondrial features of CSCs. METHODS: Colon adenocarcinoma fragments were obtained from 8 patients during surgery at Busan Paik Hospital in Korea. We used immunohistochemistry and quantitative polymerase chain reaction to compare expression of mitochondrial peroxiredoxin 3 (PRX3) in CD133(+)CD44(+) Lgr5(+)cells (CSCs) vs CD133(-)CD44(-)Lgr5(-) colon tumor cells (non-CSCs). Cell survival and expression of mitochondrial-related genes were analyzed in the presence of 5-fluorouracil and/or antimycin A. We used small-interfering and short-hairpin RNAs and an overexpression vector to study PRX3, which functions in the mitochondria. CD133(+) cells with PRX3 knockdown or overexpressing PRX3 were grown as xenograft tumors in immunocompromised mice. Metastasis was studied after injection of tumor cells in spleens of mice. We used chromatin immunoprecipitation and reporter assays to characterize transcriptional regulation of PRX3 by forkhead box protein 1. RESULTS: CSCs had a higher mitochondrial membrane potential and increased levels of adenosine triphosphate, Ca(2+), reactive oxygen species, and oxygen consumption than non-CSCs. Levels of PRX3 were increased in colon CSCs compared with non-CSCs. PRX3 knockdown reduced the viability of CSCs, but non non-CSCs, by inducing mitochondrial dysfunction. PRX3 knockdown reduced growth of CSCs as xenograft tumors or metastases in mice. The expression of FOXM1 activated transcription of PRX3 and expression of CD133 in colon CSCs. CONCLUSIONS: Human colon CSCs have increased mitochondrial function compared with colon tumor cells without stem cell properties. Colon CSCs overexpress the mitochondrial gene PRX3, which is required for maintenance of mitochondrial function and tumorigenesis, and is regulated by forkhead box protein 1, which also regulates expression of CD133 in these cells. These proteins might be therapeutic targets for colorectal cancer.

KSP inhibitor SB743921 induces death of multiple myeloma cells via inhibition of the NF-κB signaling pathway.

SB743921 is a potent inhibitor of the spindle protein kinesin and is being investigated in ongoing clinical trials for the treatment of myeloma. However, little is known about the molecular events underlying the induction of cell death in multiple myeloma (MM) by SB743921, alone or in combination treatment. Here, we report that SB743921 induces mitochondria-mediated cell death via inhibition of the NF-κB signaling pathway, but does not cause cell cycle arrest in KMS20 MM cells. SB743921-mediated inhibition of the NF-κB pathway results in reduced expression of SOD2 and Mcl-1, leading to mitochondrial dysfunction. We also found that combination treatment with SB743921 and bortezomib induces death in bortezomib-resistant KMS20 cells. Altogether, these data suggest that treatment with SB743921 alone or in combination with bortezomib offers excellent translational potential and promises to be a novel MM therapy.

Heterologous expression of IbMYB1a by different promoters exhibits different patterns of anthocyanin accumulation in tobacco.

We previously reported that the transient and stable expression of IbMYB1a produced anthocyanin pigmentation in tobacco leaves and transgenic Arabidopsis plants, respectively. To further determine the effects of different promoters on the expression of IbMYB1a and anthocyanin production, we generated and characterized stably transformed tobacco (Nicotiana tabacum SR1) plants expressing IbMYB1a under the control of three different promoters. We compared the differences in anthocyanin accumulation patterns and phenotypic features of the leaves of these transgenic tobacco plants during growth. Expression of IbMYB1a under the control of these three different promoters led to a remarkable variation in anthocyanin pigmentation in tobacco leaves. The anthocyanin contents of the leaves of the SPO-IbMYB1a-OX (SPO-M) line were higher than those of the SWPA2-IbMYB1a-OX (SPA-M) and 35S-IbMYB1a-OX (35S-M) lines. High levels of anthocyanin pigments negatively affected plant growth in the SPO-M lines, resulting delayed growth and, occasionally, a stunted phenotype. Furthermore, HPLC analysis revealed that transcriptional regulation of IbMYB1a led to the production of cyanidin-based anthocyanins in the tobacco plants. In addition, RT-PCR analysis revealed that IbMYB1a expression induced the up-regulation of several structural genes in the anthocyanin biosynthetic pathway, including DFR and ANS. Differential expression levels of IbMYB1a under the control of different promoters were highly correlated with the expression levels of the structural genes, thereby affecting anthocyanin production levels. These results indicate that IbMYB1a positively controls the expression of multiple anthocyanin biosynthetic genes and anthocyanin accumulation in heterologous tobacco plants.

The direct modulatory activity of zinc toward ion channels.

The divalent zinc ion is a cation that plays an indispensable role as a structural constituent of numerous proteins, including enzymes and transcription factors. Recently, it has been suggested that zinc also plays a dynamic role in extracellular and intracellular signaling as well. Ion channels are pore-forming proteins that control the flow of specific ions across the membrane, which is important to maintain ion gradients. In this review, we outline the modulatory effect of zinc on the activities of several ion channels through direct binding of zinc into histidine, cysteine, aspartate, and glutamate moieties of channel proteins. The binding of zinc to ion channels results in the activation or inhibition of the channel due to conformational changes. These novel aspects of ion-channel activity modulation by zinc provide new insights into the physiological regulation of ion channels.

Overexpression of the IbMYB1 gene in an orange-fleshed sweet potato cultivar produces a dual-pigmented transgenic sweet potato with improved antioxidant activity.

The R2R3-type protein IbMYB1 is a key regulator of anthocyanin biosynthesis in the storage roots of sweet potato [Ipomoea batatas (L.) Lam]. Previously, we demonstrated that IbMYB1 expression stimulated anthocyanin pigmentation in tobacco leaves and Arabidopsis. Here, we generated dual-pigmented transgenic sweet potato plants that accumulated high levels of both anthocyanins and carotenoids in a single sweet potato storage root. An orange-fleshed cultivar with high carotenoid levels was transformed with the IbMYB1 gene under the control of either the storage root-specific sporamin 1 (SPO1) promoter or the oxidative stress-inducible peroxidase anionic 2 (SWPA2) promoter. The SPO1-MYB transgenic lines exhibited higher anthocyanin levels in storage roots than empty vector control (EV) or SWPA2-MYB plants, but carotenoid content was unchanged. SWPA2-MYB transgenic lines exhibited higher levels of both anthocyanin and carotenoids than EV plants. Analysis of hydrolyzed anthocyanin extracts indicated that cyanidin and peonidin predominated in both overexpression lines. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that IbMYB1 expression in both IbMYB1 transgenic lines strongly induced the upregulation of several genes in the anthocyanin biosynthetic pathway, whereas the expression of carotenoid biosynthetic pathway genes varied between transgenic lines. Increased anthocyanin levels in transgenic plants also promoted the elevation of proanthocyanidin and total phenolic levels in fresh storage roots. Consequently, all IbMYB1 transgenic plants displayed much higher antioxidant activities than EV plants. In field cultivations, storage root yields varied between the transgenic lines. Taken together, our results indicate that overexpression of IbMYB1 is a highly promising strategy for the generation of transgenic plants with enhanced antioxidant capacity.

Production of pinostilbene compounds by the expression of resveratrol O-methyltransferase genes in Escherichia coli.

Resveratrol (3,4',5-trans-trihydroxystilbene) is a polyphenolic phytoalexin that belongs to a family of naturally occurring stilbenes. It has been reported that the health-promoting activities of certain methylated resveratrol derivatives are more effective than those of unmodified resveratrol. In this study, we isolated two candidate genes with resveratrol O-methyltransferase (ROMT) activity from grape (Vitis riparia) and sorghum (Sorghum bicolor). To assess their ROMT activities in vivo, we synthesized VrROMT and SbROMT3 following codon-optimization and expressed the VrROMTsyn and SbROMT3syn genes using a dual expression vector system. Furthermore, we attempted to produce pterostilbene from resveratrol as a substrate by the expression of two putative ROMT proteins in Escherichia coli. Unexpectedly, expression of the SbROMT3syn gene in E. coli led to the production of mono-methylated stilbene (3,4'-dihydroxy-5-methoxy-trans-stilbene, pinostilbene) from resveratrol compounds. However, a very small amount of di-methylated stilbene (3,5-dimethoxy-4'-hydroxy-trans-stilbene, pterostilbene) was also detected. Consistently, we found that in vitro methylation assays of resveratrol by recombinant SbROMT3syn produced pinostilbene as the major product besides a very small amount of pterostilbene. By contrast, very small amounts of methylated resveratrol derivatives were detected in E. coli expressing the VrROMTsyn protein. This suggests that the SbROMT3syn is more useful in the production of pinostilbene compounds than pterostilbene from resveratrol in E. coli.

Combination treatment with 2-methoxyestradiol overcomes bortezomib resistance of multiple myeloma cells.

Bortezomib is a proteasome inhibitor used for the treatment of relapsed/refractory multiple myeloma (MM). However, intrinsic and acquired resistance to bortezomib has already been observed in MM patients. In a previous report, we demonstrated that changes in the expression of mitochondrial genes lead to changes in mitochondrial activity and bortezomib susceptibility or resistance, and their combined effects contribute to the differential sensitivity or resistance of MM cells to bortezomib. Here we report that the combination treatment of bortezomib and 2-methoxyestradiol (2ME), a natural estrogen metabolite, induces mitochondria-mediated apoptotic cell death of bortezomib-resistant MM KMS20 cells via mitochondrial reactive oxygen species (ROS) overproduction. Bortezomib plus 2ME treatment induces a higher level of cell death compared with treatment with bortezomib alone and increases mitochondrial ROS and Ca(2+) levels in KMS20 cells. Pretreatment with the antioxidant N-acetyl-L-cysteine scavenges mitochondrial ROS and decreases cell death after treatment with bortezomib plus 2ME in KMS20 cells. Moreover, we observed that treatment with bortezomib plus 2ME maintains the activation of c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase kinase kinase 4/7 (MKK4/7). Collectively, combination treatment with bortezomib and 2ME induces cell death via JNK-MKK4/7 activation by overproduction of mitochondrial ROS. Therefore, combination therapy with specific mitochondrial-targeting drugs may prove useful to the development of novel strategies for the treatment of bortezomib-resistant MM patients.

Aspergillus nidulans translationally controlled tumor protein has a role in the balance between asexual and sexual differentiation and normal hyphal branching.

Translationally controlled tumor protein (TCTP) is a highly conserved and ubiquitously expressed protein present in all eukaryotes. Cellular functions of TCTP include growth promoting, allergic response and responses to various cellular stresses, but the functions in filamentous fungi have not been reported. In this report, we characterized an Aspergillus nidulans TCTP (TcpA) with high similarity to TCTP. The level of tcpa mRNA was relatively high, both during vegetative growth stage and at early phases of development. TcpA was found predominantly in the nucleus during germination and mycelial growth, and was localized in cytoplasm and nuclei of vesicles on stipes during conidia development. Deletion of tcpA resulted in abnormal hyphal branch formation during vegetative growth. The tcpA deletion inhibited sexual development, but enhanced asexual development via induction of brlA expression. These results imply that TcpA is involved in normal hyphal branch establishment during vegetative growth and also has a role in the balance between asexual and sexual differentiation.