The circRNA hsa-circ-0013561 regulates head and neck squamous cell carcinoma development via the miR-7-5p/PDK3 axis.

BACKGROUND: Circular RNAs (circRNAs) belong to a class of covalently closed single stranded RNAs that have been implicated in cancer progression. Former investigations showed that hsa-circ-0013561 is abnormally expressed in head and neck squamous cell carcinoma (HNSCC). Nevertheless, the role of hsa-circ-0013561 during the progress of HNSCC still unclear. METHODS: Present study applied FISH and qRT-PCR to examine hsa-circ-0013561 expression in HNSCC cells and tissue samples. Dual-luciferase reporter assay was employed to identify downstream targets of hsa-circ-0013561. Transwell migration, 5-ethynyl-2'-deoxyuridine incorporation, CCK8 and colony formation assays were utilized to test cell migration and proliferation. A mouse tumor xenograft model was utilized to determine the hsa-circ-0013561 roles in HNSCC progression and metastasis in vivo. RESULTS: We found that hsa-circ-0013561 was upregulated in HNSCC tissue samples. hsa-circ-0013561 downregulation inhibited HNSCC cell proliferation and migration to promote apoptosis and G1 cell cycle arrest. The dual-luciferase reporter assay revealed that miR-7-5p and PDK3 are hsa-circ-0013561 downstream targets. PDK3 overexpression or miR-7-5p suppression reversed the hsa-circ-0013561-induced silencing effects on HNSCC cell proliferation and migration. PDK3 overexpression reversed miR-7-5p-induced effects on HNSCC cell proliferation and migration. CONCLUSION: The findings suggest that hsa-circ-0013561 downregulation inhibits HNSCC metastasis and progression through PDK3 expression and miR-7-5p binding modulation.

Lycorine hydrochloride interferes with energy metabolism to inhibit chemoresistant glioblastoma multiforme cell growth through suppressing PDK3.

Glioblastoma multiforme (GBM) is the highest grade of glioma. Tumours, including GBM, possess reprogrammed metabolism, such as altered aerobic glycolysis and aberrant energy production. Lycorine hydrochloride (LH) was extracted from the bulb of Lycoris radiata. The previous study indicated that LH exerts antiviral, anti-inflammatory and antitumour effects. However, the effect of LH on GBM and the underlying molecular mechanism remain unclear. Our study revealed that LH restrained chemoresistant GBM cells growth by inhibiting PDK3 expression in vitro and in vivo. Functionally, LH inhibited the proliferation and invasive capacity of chemoresistant GBM cells in dose-dependent manner. Metabolomics and cellular energy analyses showed that LH decreased extracellular acidification rates while increased oxidative respiration and ROS levels. Mechanistically, LH inhibits the growth of GBM chemoresistant cells by regulating the expression of apoptosis-related proteins, while overexpression of of PDK3 can reverse the antitumor effect of LH. In conclusion, our study revealed that LH could reprogramme cell energy metabolism, including aerobic glycolysis suppression and oxidative phosphorylation hyperactivation by inhibiting PDK3. PDK3 may be a candidate therapeutic target for chemoresistant GBM treatment with LH.

Unveiling the clinical and electrophysiological profile of CMTX6: Insights from two Brazilian families.

BACKGROUND AND AIMS: X-linked Charcot-Marie-Tooth disease type 6 (CMTX6) is an extremely rare condition associated with mutations in the PDK3 gene. To date, only three families from different countries have been reported (Australia, South Korea, and Germany). In this study, we sought to provide a comprehensive clinical and electrophysiological characterization of two Brazilian families. METHODS: We conducted comprehensive clinical assessments, extensive electrophysiological evaluations, and performed whole-exome sequencing in the probands to investigate the genetic basis of the disease. RESULTS: Males in the family carrying the Arg162His mutation displayed early-onset motor and/or sensory axonal neuropathy, absence of tendon jerks, pes cavus, and frequently reported pain. Females in the same family exhibited a milder phenotype of the disease with later onset and some remained asymptomatic into their 50s. In the unrelated family with a single affected male, the clinical presentation was characterized by severe progressive sensorimotor polyneuropathy accompanied by neuropathic pain. INTERPRETATION: We report two Brazilian families with CMTX6 including one harboring a previously unpublished variant in the PDK3 gene, which co-segregates with the disease as expected in a X-linked disease. Notably, the clinical presentations across the five families with available descriptions, including our study, share striking similarities. Furthermore, the proximity of the three reported mutations suggests potential functional similarities and common underlying mechanisms. This study contributes to the growing knowledge of CMTX6 and underscores the importance of international collaborations in studying rare genetic disorders.

X-linked Charcot-Marie-Tooth disease type 6 (CMTX6) patients with a p.R158H mutation in the pyruvate dehydrogenase kinase isoenzyme 3 gene.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. Mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene have been found to cause X-linked dominant CMT type 6 (CMTX6). This study identified the p.R158H PDK3 mutation after screening 67 probable X-linked CMT families. The mutation fully segregated with the phenotype, and genotyping the family indicated the mutation arose on a different haplotype compared with the original Australian CMTX6 family. Results of bisulphite sequencing suggest that methylated deamination of a CpG dinucleotide may cause the recurrent p.R158H mutation. The frequency of the p.R158H PDK3 mutation in Koreans is very rare. Magnetic resonance imaging revealed fatty infiltration involving distal muscles in the lower extremities. In addition, fatty infiltrations were predominantly observed in the soleus muscles, with a lesser extent in tibialis anterior muscles. This differs from demyelinating CMT1A patients and is similar to axonal CMT2A patients. The clinical, neuroimaging, and electrophysiological findings from a second CMTX6 family with the p.R158H PDK3 mutation were similar to the axonal neuropathy reported in the Australian family.

The Therapeutic Potential of Vitamins B1, B3 and B6 in Charcot-Marie-Tooth Disease with the Compromised Status of Vitamin-Dependent Processes.

Understanding the molecular mechanisms of neurological disorders is necessary for the development of personalized medicine. When the diagnosis considers not only the disease symptoms, but also their molecular basis, treatments tailored to individual patients may be suggested. Vitamin-responsive neurological disorders are induced by deficiencies in vitamin-dependent processes. These deficiencies may occur due to genetic impairments of proteins whose functions are involved with the vitamins. This review considers the enzymes encoded by the DHTKD1, PDK3 and PDXK genes, whose mutations are observed in patients with Charcot-Marie-Tooth (CMT) disease. The enzymes bind or produce the coenzyme forms of vitamins B1 (thiamine diphosphate, ThDP) and B6 (pyridoxal-5'-phosphate, PLP). Alleviation of such disorders through administration of the lacking vitamin or its derivative calls for a better introduction of mechanistic knowledge to medical diagnostics and therapies. Recent data on lower levels of the vitamin B3 derivative, NAD+, in the blood of patients with CMT disease vs. control subjects are also considered in view of the NAD-dependent mechanisms of pathological axonal degeneration, suggesting the therapeutic potential of vitamin B3 in these patients. Thus, improved diagnostics of the underlying causes of CMT disease may allow patients with vitamin-responsive disease forms to benefit from the administration of the vitamins B1, B3, B6, their natural derivatives, or their pharmacological forms.

Human Lung Cancer (A549) Cell Line Cytotoxicity and Anti-Leishmania major Activity of Carissa macrocarpa Leaves: A Study Supported by UPLC-ESI-MS/MS Metabolites Profiling and Molecular Docking.

Lung cancer and cutaneous leishmaniasis are critical diseases with a relatively higher incidence in developing countries. In this research, the activity of Carissa macrocarpa leaf hydromethanolic extract and its solvent-fractions (n-hexane, EtOAc, n-butanol, and MeOH) against the lung adenocarcinoma cell line (A549) and Leishmania major was investigated. The MeOH fraction exhibited higher cytotoxic activity (IC50 1.57 ± 0.04 µg/mL) than the standard drug, etoposide (IC50 50.8 ± 3.16 µg/mL). The anti-L. major results revealed strong growth inhibitory effects of the EtOAc fraction against L. major promastigotes (IC50 27.52 ± 0.7 µg/mL) and axenic amastigotes (29.33 ± 4.86% growth inhibition at 100 µg/mL), while the butanol fraction exerted moderate activity against promastigotes (IC50 73.17 ± 1.62), as compared with miltefosine against promastigotes (IC50 6.39 ± 0.29 µg/mL) and sodium stibogluconate against axenic amastigotes (IC50 22.45 ± 2.22 µg/mL). A total of 102 compounds were tentatively identified using UPLC-ESI-MS/MS analysis of the total extract and its fractions. The MeOH fraction was found to contain several flavonoids and flavan-3-ol derivatives with known cytotoxic properties, whereas the EtOAc fractions contained triterpene, hydroxycinnamoyl, sterol, and flavanol derivatives with known antileishmanial activity. Molecular docking of various polyphenolics of the MeOH fraction with HDAC6 and PDK3 enzymes demonstrates high binding affinity of the epicatechin 3-O-ß-D-glucopyranoside and catechin-7-O-ß-D-glucopyranoside toward HDAC6, and procyanidin C2, procyanidin B5 toward PDK3. These results are promising and encourage the pursuit of preclinical research using C. macrocarpa's MeOH fraction as anti-lung cancer and the EtOAc fraction as an anti-L. major drug candidates.

LncRNA POT1-AS1 accelerates the progression of gastric cancer by serving as a competing endogenous RNA of microRNA-497-5p to increase PDK3 expression.

BACKGROUND: Gastric cancer (GC) is the most common malignant tumor of the digestive system. Although progress has been reported in terms of treatment, it is still the second leading cause of cancer-related death. Long non-coding RNAs have been shown to play a key role in human cancers in recent investigations. However, the role of POT1-AS1 in GC is still unclear. METHODS: The relative POT1-AS1 level in GC tissues and paracancerous tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The biological function of POT1-AS1 was studied by CCK8 and Transwell assays in vitro experiments. Moreover, the downstream target genes of POT1-AS1 were predicted by bioinformatics. RESULTS: In this study, high POT1-AS1 expression in GC cells was confirmed, and its elevated expression was linked to patients' negative clinicopathological characteristics, as well as shorter disease-free survival (DFS) and overall survival (OS). POT1-AS1 was shown to serve as a competing endogenous RNA (ceRNA) by sponging miR-497-5p to increase PDK3 expression. The impact of POT1-AS1 silencing on GC malignant phenotypes could be reversed by suppressing miR-497-5p or restoring PDK3, according to rescue experiments. CONCLUSIONS: In brief, POT1-AS1 acted as an oncogenic lncRNA in GC, facilitating GC development by affecting the miR-497-5p/PDK3 axis, implying that the POT1-AS1/miR-497-5p/PDK3 axis is a useful target in anticancer therapy.

Metabolic enzyme PDK3 forms a positive feedback loop with transcription factor HSF1 to drive chemoresistance.

Background & Aims: Dysregulation of metabolism plays an important role in the development and progression of cancers, while the underlying mechanisms remain largely unknown. This study aims to explore the regulation and relevance of glycolysis in chemoresistance of gastric cancer. Methods: Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by metabolism profiling, microarray gene expression, PCR or western blotting. Cancer cell growth in vitro or in vivo were analyzed by viability, apoptosis and nude mice assay. Immunoprecipation was used to explore the interaction of proteins with other proteins or DNAs. Results: By metabolic and gene expression profiling, we found that pyruvate dehydrogenase kinase 3 (PDK3) was highly expressed to promote glycolysis in chemoresistant cancer cells. Its genetic or chemical inhibition reverted chemoresistance in vitro and in vivo. It was transcriptionally regulated by transcription factor HSF1 (Heat shock factor 1). Interestingly, PDK3 can localize in the nucleus and interact with HSF1 to disrupt its phosphorylation by GSK3ß. Since HSF1 was subjected to FBXW7-catalyzed polyubiquitination in a phosphorylation-dependent manner, PDK3 prevented HSF1 from proteasomal degradation. Thus, metabolic enzyme PDK3 and transcription factor HSF1 forms a positive feedback loop to promote glycolysis. As a result, inhibition of HSF1 impaired enhanced glycolysis and reverted chemoresistance both in vitro and in vivo. Conclusions: PDK3 forms a positive feedback loop with HSF1 to drive glycolysis in chemoresistance. Targeting this mitonuclear communication may represent a novel approach to overcome chemoresistance.

The lncRNA-DLEU2/miR-186-5p/PDK3 axis promotes the progress of glioma cells.

Long non-coding RNAs (lncRNAs) have great value in research on tumour targeted therapy, including for glioma. In the present study, we investigated the role of the lncRNA deleted in lymphocytic leukaemia 2 (lncRNA-DLEU2) in glioma. First, we found that lncRNA-DLEU2 is highly expressed in glioma tissues and cell lines. Next, experiments in cells showed that lncRNA-DLEU2 knockdown inhibited, whereas lncRNA-DLEU2 overexpression promoted, the clone formation, migration and invasion of glioma cells. A luciferase reporter assay and an RNA immunoprecipitation assay demonstrated that lncRNA-DLEU2 acts as a sponge for miR-186-5p in glioma cells. Further, studies suggested that miR-186-5p inhibits the expression of PDK3, which is an oncogene in glioma. Moreover, with rescue experiments, we demonstrated that lncRNA-DLEU2 regulates the expression of PDK3 and the progression of glioma in a miR-186-5p-dependent manner. Finally, we also showed that lncRNA-DLEU2 promotes glioma growth in a manner that is related to miR-186-5p and PDK3 in vivo. In conclusion, our study reported for the first time that lncRNA-DLEU2 promotes glioma progression by targeting the miR-186-5p/PDK3 axis. These findings provide novel strategies for the gene therapy treatment of glioma.

miR-497-5p inhibits gastric cancer cell proliferation and growth through targeting PDK3.

MicroRNA plays an important role in gastric cancer (GC) development, while the function of miR-497-5p in this disease remains unknown. In the present study, we demonstrated miR-497-5p as a tumor suppressive microRNA in GC. miR-497-5p was down-regulated in GC tissues and its expression was associated with the disease stage. Inhibition of miR-497-5p promoted GC cell proliferation and growth. By contrast, miR-497-5p ectopic expression suppressed the proliferation and growth of GC cells. In addition, miR-497-5p inhibited DNA synthesis and enhanced apoptosis in GC cells. The cell cycle progression was suppressed by miR-497-5p. Mechanistically, miR-497-5p directly targeted and suppressed the expression of pyruvate dehydrogenase kinase 3 (PDK3), which is highly expressed in GC tissues. Over-expression of PDK3 promoted the proliferation of GC cells. Our study revealed that miR-497-5p inhibited GC cell proliferation and growth via targeting PDK3.

Investigation of inhibitory potential of quercetin to the pyruvate dehydrogenase kinase 3: Towards implications in anticancer therapy.

Pyruvate dehydrogenase kinase 3 (PDK3) is a mitochondrial protein, has recently been considered as a potential pharmacological target for varying types of cancer. Here, we report the binding mechanism of quercetin to the PDK3 by using molecular docking, simulation, fluorescence spectroscopy and isothermal titration calorimetric assays. Molecular docking along with simulation provided an in-depth analysis of protein-ligand interactions. We have observed that quercetin interacts to the important residues of active site cavity of PDK3 and shows a well-ordered conformational fitting. The stability of quercetin-PDK3 complex is maintained by several non-covalent interactions throughout the simulation. To complement in silico findings with the experiments, we have successfully expressed and purified human PDK3. Both fluorescence and isothermal titration calorimetric experiments showed excellent binding affinity of quercetin to the PDK3. Kinase inhibition assay further revealed a significant inhibitory potential of quercetin to the PDK3 with the IC50 values in µM range. Quercetin is non-toxic to HEK293, and significantly inhibits the proliferation of cancer (HepG2 and A549) cell lines. All these observations clearly indicate that quercetin may be further evaluated as promising therapeutic molecule for PDK3 with required modifications and in vivo validation.

Silencing of NAC1 Expression Induces Cancer Cells Oxidative Stress in Hypoxia and Potentiates the Therapeutic Activity of Elesclomol.

In order to survive under conditions of low oxygen, cancer cells can undergo a metabolic switch to glycolysis and suppress mitochondrial respiration in order to reduce oxygen consumption and prevent excessive amounts of reactive oxygen species (ROS) production. Nucleus accumbens-1 (NAC1), a nuclear protein of the BTB/POZ gene family, has pivotal roles in cancer development. Here, we identified that NAC1-PDK3 axis as necessary for suppression of mitochondrial function, oxygen consumption, and more harmful ROS generation and protects cancer cells from apoptosis in hypoxia. We show that NAC1 mediates suppression of mitochondrial function in hypoxia through inducing expression of pyruvate dehydrogenase kinase 3 (PDK3) by HIF-1α at the transcriptional level, thereby inactivating pyruvate dehydrogenase and attenuating mitochondrial respiration. Re-expression of PDK3 in NAC1 absent cells rescued cells from hypoxia-induced metabolic stress and restored the activity of glycolysis in a xenograft mouse model, and demonstrated that silencing of NAC1 expression can enhance the antitumor efficacy of elesclomol, a pro-oxidative agent. Our findings reveal a novel mechanism by which NAC1 facilitates oxidative stress resistance during cancer progression, and chemo-resistance in cancer therapy.

Auxin as an inducer of asymmetrical division generating the subsidiary cells in stomatal complexes of Zea mays.

The data presented in this work revealed that in Zea mays the exogenously added auxins indole-3-acetic acid (IAA) and 1-napthaleneacetic acid (NAA), promoted the establishment of subsidiary cell mother cell (SMC) polarity and the subsequent subsidiary cell formation, while treatment with auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and 1-napthoxyacetic acid (NOA) specifically blocked SMC polarization and asymmetrical division. Furthermore, in young guard cell mother cells (GMCs) the PIN1 auxin efflux carriers were mainly localized in the transverse GMC faces, while in the advanced GMCs they appeared both in the transverse and the lateral ones adjacent to SMCs. Considering that phosphatidyl-inositol-3-kinase (PI3K) is an active component of auxin signal transduction and that phospholipid signaling contributes in the establishment of polarity, treatments with the specific inhibitor of the PI3K LY294002 were carried out. The presence of LY294002 suppressed polarization of SMCs and prevented their asymmetrical division, whereas combined treatment with exogenously added NAA and LY294002 restricted the promotional auxin influence on subsidiary cell formation. These findings support the view that auxin is involved in Z. mays subsidiary cell formation, probably functioning as inducer of the asymmetrical SMC division. Collectively, the results obtained from treatments with auxin transport inhibitors and the appearance of PIN1 proteins in the lateral GMC faces indicate a local transfer of auxin from GMCs to SMCs. Moreover, auxin signal transduction seems to be mediated by the catalytic function of PI3K.