Temperature induces significant changes in both glycolytic reserve and mitochondrial spare respiratory capacity in colorectal cancer cell lines.

Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, microplate based, label-free detection approach. This study investigates how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy.

Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development.

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

Genetic impairment of succinate metabolism disrupts bioenergetic sensing in adrenal neuroendocrine cancer.

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

Microtubule-Based Mitochondrial Dynamics as a Valuable Therapeutic Target in Cancer.

Mitochondria constitute an ever-reorganizing dynamic network that plays a key role in several fundamental cellular functions, including the regulation of metabolism, energy production, calcium homeostasis, production of reactive oxygen species, and programmed cell death. Each of these activities can be found to be impaired in cancer cells. It has been reported that mitochondrial dynamics are actively involved in both tumorigenesis and metabolic plasticity, allowing cancer cells to adapt to unfavorable environmental conditions and, thus, contributing to tumor progression. The mitochondrial dynamics include fusion, fragmentation, intracellular trafficking responsible for redistributing the organelle within the cell, biogenesis, and mitophagy. Although the mitochondrial dynamics are driven by the cytoskeleton-particularly by the microtubules and the microtubule-associated motor proteins dynein and kinesin-the molecular mechanisms regulating these complex processes are not yet fully understood. More recently, an exchange of mitochondria between stromal and cancer cells has also been described. The advantage of mitochondrial transfer in tumor cells results in benefits to cell survival, proliferation, and spreading. Therefore, understanding the molecular mechanisms that regulate mitochondrial trafficking can potentially be important for identifying new molecular targets in cancer therapy to interfere specifically with tumor dissemination processes.

Intrinsic OXPHOS limitations underlie cellular bioenergetics in leukemia.

Currently there is great interest in targeting mitochondrial oxidative phosphorylation (OXPHOS) in cancer. However, notwithstanding the targeting of mutant dehydrogenases, nearly all hopeful 'mito-therapeutics' cannot discriminate cancerous from non-cancerous OXPHOS and thus suffer from a limited therapeutic index. Using acute myeloid leukemia (AML) as a model, herein, we leveraged an in-house diagnostic biochemical workflow to identify 'actionable' bioenergetic vulnerabilities intrinsic to cancerous mitochondria. Consistent with prior reports, AML growth and proliferation was associated with a hyper-metabolic phenotype which included increases in basal and maximal respiration. However, despite having nearly 2-fold more mitochondria per cell, clonally expanding hematopoietic stem cells, leukemic blasts, as well as chemoresistant AML were all consistently hallmarked by intrinsic OXPHOS limitations. Remarkably, by performing experiments across a physiological span of ATP free energy, we provide direct evidence that leukemic mitochondria are particularly poised to consume ATP. Relevant to AML biology, acute restoration of oxidative ATP synthesis proved highly cytotoxic to leukemic blasts, suggesting that active OXPHOS repression supports aggressive disease dissemination in AML. Together, these findings argue against ATP being the primary output of leukemic mitochondria and provide proof-of-principle that restoring, rather than disrupting, OXPHOS may represent an untapped therapeutic avenue for combatting hematological malignancy and chemoresistance.

Deceptology in cancer and vaccine sciences: Seeds of immune destruction-mini electric shocks in mitochondria: Neuroplasticity-electrobiology of response profiles and increased induced diseases in four generations - A hypothesis.

From Rockefeller's support of patent medicine to Gates' patent vaccines, medical establishment invested a great deal in intellectual ignorance. Through the control over medical education and research it has created a public illusion to prop up corporate profit and encouraged the lust for money and power. An overview of data on cancer and vaccine sciences, the status of Americans' health, a survey of repeated failed projects, economic toxicity, and heavy drug consumption or addiction among young and old provide compelling evidence that in the twentieth century nearly all classic disease categories (congenital, inheritance, neonatal, or induced) shifted to increase induced diseases. Examples of this deceptology in ignoring or minimizing, and mocking fundamental discoveries and theories in cancer and vaccine sciences are attacks on research showing that (a), effective immunity is responsible for defending and killing pathogens and defective cancerous cells, correcting and repairing genetic mutations; (b) viruses cause cancer; and (c), abnormal gene mutations are often the consequences of (and secondary to) disturbances in effective immunity. The outcomes of cancer reductionist approaches to therapies reveal failure rates of 90% (+/-5) for solid tumors; loss of over 50 million lives and waste of $30-50 trillions on too many worthless, out-of-focus, and irresponsible projects. Current emphasis on vaccination of public with pathogen-specific vaccines and ingredients seems new terms for drugging young and old. Cumulative exposures to low level carcinogens and environmental hazards or high energy electronic devices (EMF; 5G) are additional triggers to vaccine toxicities (antigen-mitochondrial overload) or "seeds of immune destruction" that create mini electrical shocks (molecular sinks holes) in highly synchronized and regulated immune network that retard time-energy-dependent biorhythms in organs resulting in causes, exacerbations or consequences of mild, moderate or severe immune disorders. Four generations of drug-dependent Americans strongly suggest that medical establishment has practiced decades of intellectual deception through its claims on "war on cancer"; that cancer is 100, 200, or 1000 diseases; identification of "individual" genetic mutations to cure diseases; "vaccines are safe". Such immoral and unethical practices, along with intellectual harassment and bullying, censoring or silencing of independent and competent professionals ("Intellectual Me Too") present grave concerns, far greater compared with the sexual harassment of 'Me Too' movement that was recently spearheaded by NIH. The principal driving forces behind conducting deceptive and illogical medical/cancer and vaccine projects seem to be; (a) huge return of investment and corporate profit for selling drugs and vaccines; (b) maintenance of abusive power over public health; (c) global control of population growth via increased induction of diseases, infertility, decline in life-span, and death. An overview of accidental discoveries that we established and extended since 1980s, on models of acute and chronic ocular inflammatory diseases, provides series of the first evidence for a direct link between inflammation and multistep immune dysfunction in tumorigenesis and angiogenesis. Results are relevant to demonstrate that current emphasis on vaccinating the unborn, newborn, or infant would induce immediate or long-term immune disorders (eg, low birth weight, preterm birth, fatigue, autism, epilepsy/seizures, BBB leakage, autoimmune, neurodegenerative or digestive diseases, obesity, diabetes, cardiovascular problems, or cancers). Vaccination of the unborn is likely to disturb trophoblast-embryo-fetus-placenta biology and orderly growth of embryo-fetus, alter epithelial-mesenchymal transition or constituent-inducible receptors, damage mitochondria, and diverse function of histamine-histidine pathways. Significant increased in childhood illnesses are likely due to toxicities of vaccine and incipient (eg, metals [Al, Hg], detergents, fetal tissue, DNA/RNA) that retard bioenergetics of mitochondria, alter polarization-depolarization balance of tumoricidal (Yin) and tumorigenic (Yang) properties of immunity. Captivated by complex electobiology of immunity, this multidisciplinary perspective is an attempt to initiate identifying bases for increased induction of immune disorders in three to four generations in America. We hypothesize that (a) gene-environment-immune biorhythms parallel neuronal function (brain neuroplasticity) with super-packages of inducible (adaptive or horizontal) electronic signals and (b) autonomic sympathetic and parasympathetic circuitry that shape immunity (Yin-Yang) cannot be explained by limited genomics (innate, perpendicular) that conventionally explain certain inherited diseases (eg, sickle cell anemia, progeria). Future studies should focus on deep learning of complex electrobiology of immunity that requires differential bioenergetics from mitochondria and cytoplasm. Approaches to limit or control excessive activation of gene-environment-immunity are keys to assess accurate disease risk formulations, prevent inducible diseases, and develop universal safe vaccines that promote health, the most basic human right.

MicroRNA regulation of cancer metabolism: role in tumour suppression.

Mitochondria are critical regulators of cell metabolism; thus, mitochondrial dysfunction is associated with many metabolic disorders, including cancer. Altered metabolism is a common property of cancer cells that exhibit enhanced capacity to 'ferment' glucose to pyruvate and then lactate, even in the presence of sufficient oxygen to support mitochondrial metabolism. Recently, it was reported that microRNAs (miRNAs) regulate important signalling pathways in mitochondria and many of these miRNAs are deregulated in various cancers. Different regulatory mechanisms can control miRNA expression at the genetic or epigenetic level, thus affecting the biogenetic machinery via recruitment of specific transcription factors. Metabolic reprogramming that cancer cells undergo during tumorigenesis offers a wide range of potential targets to impair tumour progression. MiRNAs participate in controlling cancer cell metabolism by regulating the expression of genes whose protein products either directly regulate metabolic machinery or indirectly modulate the expression of metabolic enzymes, serving as master regulators. Thus, modulation of the level of miRNAs may provide a new approach for the treatment of neoplastic diseases.