Melatonin and Pathological Cell Interactions: Mitochondrial Glucose Processing in Cancer Cells.

Melatonin is synthesized in the pineal gland at night. Since melatonin is produced in the mitochondria of all other cells in a non-circadian manner, the amount synthesized by the pineal gland is less than 5% of the total. Melatonin produced in mitochondria influences glucose metabolism in all cells. Many pathological cells adopt aerobic glycolysis (Warburg effect) in which pyruvate is excluded from the mitochondria and remains in the cytosol where it is metabolized to lactate. The entrance of pyruvate into the mitochondria of healthy cells allows it to be irreversibly decarboxylated by pyruvate dehydrogenase (PDH) to acetyl coenzyme A (acetyl-CoA). The exclusion of pyruvate from the mitochondria in pathological cells prevents the generation of acetyl-CoA from pyruvate. This is relevant to mitochondrial melatonin production, as acetyl-CoA is a required co-substrate/co-factor for melatonin synthesis. When PDH is inhibited during aerobic glycolysis or during intracellular hypoxia, the deficiency of acetyl-CoA likely prevents mitochondrial melatonin synthesis. When cells experiencing aerobic glycolysis or hypoxia with a diminished level of acetyl-CoA are supplemented with melatonin or receive it from another endogenous source (pineal-derived), pathological cells convert to a more normal phenotype and support the transport of pyruvate into the mitochondria, thereby re-establishing a healthier mitochondrial metabolic physiology.

Zhoushi Qi Ling decoction represses docetaxel resistance and glycolysis of castration-resistant prostate cancer via regulation of SNHG10/miR-1271-5p/TRIM66 axis.

Docetaxel resistance developed in half of castration-resistant prostate cancer (CRPC) patients hinders its long-term clinical application. The current study was designed to investigate the effects of Chinese medicine Zhoushi Qi Ling decoction on the docetaxel resistance of prostate cancer as well as elucidate the underlying molecular mechanism. In our study, Qi Ling significantly decreased viability and colony formation as well as increased apoptosis of docetaxel-resistant (DR) CRPC cells. Qi Ling-treated DR cells exhibited decreased glucose consumption, lactate release and pyruvate production. Moreover, lncRNA SNHG10 was upregulated in DR tissues of CRPC patients and was negatively correlated with the progression-free survival. Bioinformatics analysis indicated miR-1271-5p as the associated miRNA possibly binding with SNHG10. miR-1271-5p up-regulation dramatically decreased the luciferase activity of SNHG10 in DR cells. SNHG10 knockdown sharply increased the expression of miR1271-5p in DR cells. Targetscan predicted TRIM66 as one of the downstream targets of miR-1271-5p. miR-1271-5p up-regulation drastically reduced luciferase activity as well as TRIM66 expression in DR cells. Also, the knockdown of SNHG10 remarkably suppressed the expression of TRIM66 in DR cells. Additionally, Qi Ling treatment reduced SNHG10 and TRIM66, while increased miR1271-5p, in DR cells. In summary, Qi Ling inhibited docetaxel resistance and glycolysis of CRPC possibly via SNHG10/miR-1271-5p/TRIM66 pathway.

The pathways related to glutamine metabolism, glutamine inhibitors and their implication for improving the efficiency of chemotherapy in triple-negative breast cancer.

Breast cancer (BC) is a heterogeneous cancer with multiple subtypes affecting women worldwide. Triple-negative breast cancer (TNBC) is a prominent subtype of BC with poor prognosis and an aggressive phenotype. Recent understanding of metabolic reprogramming supports its role in the growth of cancer cells and their adaptation to their microenvironment. The Warburg effect is characterized by the shift from oxidative to reductive metabolism and external secretion of lactate. The Warburg effect prevents the use of the required pyruvate in the tricarboxylic acid (TCA) cycle progressing through pyruvate dehydrogenase inactivation. Therefore, it is a major regulatory mechanism to promote glycolysis and disrupt the TCA cycle. Glutamine (Gln) can supply the complementary energy for cancer cells. Additionally, it is the main substrate to support bioenergetics and biosynthetic activities in cancer cells and plays a vital role in a wide array of other processes such as ferroptosis. Thus, the switching of glucose to Gln in the TCA cycle toward reductive Gln metabolism is carried out by hypoxia-inducible factors (HIFs) conducted through the Warburg effect. The literature suggests that the addiction of TNBC to Gln could facilitate the proliferation and invasiveness of these cancers. Thus, Gln metabolism inhibitors, such as CB-839, could be applied to manage the carcinogenic properties of TNBC. Such inhibitors, along with conventional chemotherapy agents, can potentially improve the efficiency and efficacy of TNBC treatment. In this review, we discuss the associations between glucose and Gln metabolism and control of cancer cell growth from the perspective that Gln metabolism inhibitors could improve the current chemotherapy drug effects.

Sublethal heat stress-induced O-GlcNAcylation coordinates the Warburg effect to promote hepatocellular carcinoma recurrence and metastasis after thermal ablation.

The malignant transformation of residual hepatocellular carcinoma (HCC) cells after thermal ablation is considered as the main factor promoting postoperative HCC progression, which greatly limits the improvement of long-term survival, and at present there is no effective targeted therapeutic strategies. The Warburg effect is a metabolic feature correlated highly with malignant transformation (e.g. epithelial-to-mesenchymal transition [EMT]). Here, we showed that sublethal heat stress triggered a stronger Warburg effect of HCC cells, which contributed to the thermotolerance and invasion of HCC cells. Sublethal heat stress-induced O-GlcNAcylation was involved in this process. Such enhanced Warburg effect in HCC cells may be eliminated through O-GlcNAcylation inhibition, resulting in impaired thermotolerance and EMT, and thereby preventing tumor recurrence and metastasis of HCC-bearing mice after insufficient thermal ablation. Finally, we present evidence that sublethal heat stress-induced O-GlcNAcylation regulates the Warburg effect in HCC cells by promoting hypoxia-inducible factor 1α (HIF-1α) stability. In conclusion, the present study suggests that O-GlcNAcylation coordinates the Warburg effect to promote HCC progression after thermal ablation, which may serve as a novel potential target for controlling postoperative HCC recurrence and metastasis.

Shikonin inhibits the Warburg effect, cell proliferation, invasion and migration by downregulating PFKFB2 expression in lung cancer.

Lung cancer is one of the most lethal diseases and therefore poses a significant threat to human health. The Warburg effect, which is the observation that cancer cells predominately produce energy through glycolysis, even under aerobic conditions, is a hallmark of cancer. 6­phosphofructo­2­kinase/fructose­2,6­biphosphatase 2 (PFKFB) is an important regulator of glycolysis. Shikonin is a Traditional Chinese herbal medicine, which has been reported to exert antitumor effects. The present study aimed to investigate the anticancer activity of shikonin in lung cancer. Cell Counting Kit­8 (CCK­8) and colony formation assays were used to analyze proliferation in A549 and H446 cells. Wound healing and Transwell assays were used to measure migration and invasion in A549 and H446 cells. Cell apoptosis was analyzed using flow cytometry. Lactate levels, glucose uptake and cellular ATP levels were measured using their corresponding commercial kits. Western blotting was performed to analyze the protein expression levels of key enzymes involved in aerobic glucose metabolism. Reverse transcription­quantitative PCR was used to analyze the mRNA expression levels of PFKFB2. The results of the present study revealed that PFKFB2 expression levels were significantly upregulated in NSCLC tissues. Shikonin treatment decreased the proliferation, migration, invasion, glucose uptake, lactate levels, ATP levels and PFKFB2 expression levels and increased apoptosis in lung cancer cells in a dose­dependent manner. The overexpression of PFKFB2 increased the proliferation, migration, glucose uptake, lactate levels and ATP levels in lung cancer cells, while the knockdown of PFKFB2 expression exerted the opposite effects. Moreover, there were no significant differences in lung cancer cell migration, apoptosis, glucose uptake, lactate levels and ATP levels between cells with knocked down PFKFB2 expression or treated with shikonin and the knockdown of PFKFB2 in cells treated with shikonin. In conclusion, the results of the present study revealed that shikonin inhibited the Warburg effect and exerted antitumor activity in lung cancer cells, which was associated with the downregulation of PFKFB2 expression.

In Vivo Anticancer Activity of AZD3965: A Systematic Review.

Proliferating cancer cells have high energy demands, which is mainly obtained through glycolysis. The transmembrane trafficking of lactate, a major metabolite produced by glycolytic cancer cells, relies on monocarboxylate transporters (MCTs). MCT1 optimally imports lactate, although it can work bidirectionally, and its activity has been linked to cancer aggressiveness and poor outcomes. AZD3965, a specific MCT1 inhibitor, was tested both in vitro and in vivo, with encouraging results; a phase I clinical trial has already been undertaken. Thus, analysis of the experimental evidence using AZD3965 in different cancer types could give valuable information for its clinical use. This systematic review aimed to assess the in vivo anticancer activity of AZD3965 either alone (monotherapy) or with other interventions (combination therapy). Study search was performed in nine different databases using the keywords "AZD3965 in vivo" as search terms. The results show that AZD3965 successfully decreased tumor growth and promoted intracellular lactate accumulation, which confirmed its effectiveness, especially in combined therapy. These results support the setup of clinical trials, but other important findings, namely AZD3965 enhanced activity when given in combination with other therapies, or MCT4-induced treatment resistance, should be further considered in the clinical trial design to improve therapy response.

ITGB2-mediated metabolic switch in CAFs promotes OSCC proliferation by oxidation of NADH in mitochondrial oxidative phosphorylation system.

Objectives: Integrins, the coordinator of extracellular and intracellular signaling, are often found to be aberrant in tumors and can reshape the tumor microenvironment. Although previous studies showed that integrin beta 2 (ITGB2) is important for host defense, its expression profile and role in tumors, especially in cancer associated fibroblasts (CAFs) are still unknown. Methods: Immunofluorescence stain and fluorescence activated cell sorting were used to analyze the ITGB2 expression profile in oral squamous cell carcinoma (OSCC). RT-PCR and western blot were used to compare ITGB2 expression in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs). Clinical data and function-based experiments were used to investigate the promoting tumor growth ability of ITGB2 expressing CAFs. Enhanced glycolysis activity was identified by using bioinformatics analyses and GC/MS assays. MCT1 knockdown OSCC cell lines were constructed to explore the pro-proliferative mechanisms of ITGB2 expressing CAFs in multiple in vitro and in vivo assays. Results: We found that CAFs exhibited significantly higher ITGB2 expression than the matched NFs. In addition, higher ITGB2 expression in CAFs was correlated with higher TNM stages and more Ki67+ tumor cells, indicating its ability to promote OSCC proliferation. Further, co-culture assay demonstrated that ITGB2-mediated lactate release in CAFs promoted OSCC cell proliferation. Mechanically, ITGB2 regulated PI3K/AKT/mTOR pathways to enhance glycolysis activity in CAFs. Accordingly, lactate derived from ITGB2-expressing CAFs was absorbed and metabolized in OSCC to generate NADH, which was then oxidized in the mitochondrial oxidative phosphorylation system (OXPHOS) to produce ATP. Notably, inhibiting the OXPHOS system with metformin delayed the proliferative capacity of OSCC cells cultured in the ITGB2-expressing CAFs medium. Conclusions: Our study uncovered the ITGB2high pro-tumoral CAFs that activated the PI3K/AKT/mTOR axis to promote tumor proliferation in OSCC by NADH oxidation in the mitochondrial oxidative phosphorylation system.

Effects and mechanisms of fatty acid metabolism­mediated glycolysis regulated by betulinic acid­loaded nanoliposomes in colorectal cancer.

Although previous studies have demonstrated that triterpenoids, such as betulinic acid (BA), can inhibit tumor cell growth, their potential targets in colorectal cancer (CRC) metabolism have not been systematically investigated. In the present study, BA­loaded nanoliposomes (BA­NLs) were prepared, and their effects on CRC cell lines were evaluated. The aim of the present study was to determine the anticancer mechanisms of action of BA­NLs in fatty acid metabolism­mediated glycolysis, and investigate the role of key targets, such as acyl­CoA synthetase (ACSL), carnitine palmitoyltransferase (CPT) and acetyl CoA, in promoting glycolysis, which is activated by inducing hexokinase (HK), phosphofructokinase­1 (PFK­1), phosphoenolpyruvate (PEP) and pyruvate kinase (PK) expression. The results demonstrated that BA­NLs significantly suppressed the proliferation and glucose uptake of CRC cells by regulating potential glycolysis and fatty acid metabolism targets and pathways, which forms the basis of the anti­CRC function of BA­NLs. Moreover, the effects of BA­NLs were further validated by demonstrating that the key targets of HK2, PFK­1, PEP and PK isoenzyme M2 (PKM2) in glycolysis, and of ACSL1, CPT1a and PEP in fatty acid metabolism, were blocked by BA­NLs, which play key roles in the inhibition of glycolysis and fatty acid­mediated production of pyruvate and lactate. The results of the present study may provide a deeper understanding supporting the hypothesis that liposomal BA may regulate alternative metabolic pathways implicated in CRC adjuvant therapy.

Mito-oncology agent: fermented extract suppresses the Warburg effect, restores oxidative mitochondrial activity, and inhibits in vivo tumor growth.

Mitochondrial dysfunction and significant changes in metabolic pathways accompany cancer development and are responsible for maintaining the tumor microenvironment. Normal mitochondria can trigger intrinsic apoptosis by releasing cytochrome c into the cytosol. The survival of malignant cells highly depends on the suppression of this function. We validated that A250, a highly purified fraction of fermented wheat germ extract (FWGE), increases the carbon flux into the mitochondria, the expression of key elements of the Krebs cycle and oxidative phosphorylation (OXPHOS). The increased respiratory chain activity is related to the mitochondria's ability to release cytochrome c into the cytosol, which triggers the apoptotic cascade. The 68% tumor growth inhibitory effect observed in the murine melanoma study is related to this effect, as proteomic analysis validated similar changes in mitochondrial protein levels in the isolated tumor tissue samples. Blood count data indicated that this effect was not accompanied by general toxicity. This study is significant, as it shows that a highly concentrated form of FWGE is an effective agent that increases normal mitochondrial functionality. The lack of hepatotoxic and general toxic effects makes A250 an excellent candidate targeting mitochondria function in cancer therapy.

Therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and neuroblastoma progression.

As a hallmark of metabolic reprogramming, aerobic glycolysis contributes to tumorigenesis and aggressiveness. However, the mechanisms and therapeutic strategies regulating aerobic glycolysis in neuroblastoma (NB), one of leading causes of cancer-related death in childhood, still remain elusive. Methods: Transcriptional regulators and their downstream glycolytic genes were identified by a comprehensive screening of publicly available datasets. Dual-luciferase, chromatin immunoprecipitation, real-time quantitative RT-PCR, western blot, gene over-expression or silencing, co-immunoprecipitation, mass spectrometry, peptide pull-down assay, sucrose gradient sedimentation, seahorse extracellular flux, MTT colorimetric, soft agar, matrigel invasion, and nude mice assays were undertaken to explore the biological effects and underlying mechanisms of transcriptional regulators in NB cells. Survival analysis was performed by using log-rank test and Cox regression assay. Results: Transcription factor myeloid zinc finger 1 (MZF1) was identified as an independent prognostic factor (hazard ratio=2.330, 95% confidence interval=1.021 to 3.317), and facilitated glycolysis process through increasing expression of hexokinase 2 (HK2) and phosphoglycerate kinase 1 (PGK1). Meanwhile, a 21-amino acid peptide encoded by upstream open reading frame of MZF1, termed as MZF1-uPEP, bound to zinc finger domain of Yin Yang 1 (YY1), resulting in repressed transactivation of YY1 and decreased transcription of MZF1 and downstream genes HK2 and PGK1. Administration of a cell-penetrating MZF1-uPEP or lentivirus over-expressing MZF1-uPEP inhibited the aerobic glycolysis, tumorigenesis and aggressiveness of NB cells. In clinical NB cases, low expression of MZF1-uPEP or high expression of MZF1, YY1, HK2, or PGK1 was associated with poor survival of patients. Conclusions: These results indicate that therapeutic targeting of YY1/MZF1 axis by MZF1-uPEP inhibits aerobic glycolysis and NB progression.