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Toxicology Research Jul 2020Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with...
Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with observations in man. As emphasized by the '3Rs' and the desire to phase-out animal research, models are needed. One potential approach uses clinostat-cultured 3D HepG2-C3A liver-mimetic spheroids. They take 18 days to recover physiological functionality and reach a metabolic equilibrium, which is thereafter stable for a year. Acute and chronic repeated-dose studies of six drugs (amiodarone, diclofenac, metformin, phenformin, paracetamol and valproic acid) suggest that spheroids are more predictive of human toxicity than either 2D-cultured HepG2 cells or primary human hepatocytes. Repeated non-lethal treatment results in a clear response and return to equilibrium. Mitochondrial toxic compounds can be identified using a galactose-based medium. Some drugs induced a protective (or stress) response that intensifies after the second treatment. This 3D spheroid model is inexpensive, highly reproducible and well-suited for the determination of repeated-dose toxicity of compounds (naturally or chemically synthesized).
PubMed: 32905230
DOI: 10.1093/toxres/tfaa033 -
RSC Advances Apr 2021A bar micro-solid phase (bar μ-SPE) extraction method using either graphene or zeolite or their mixtures as an adsorbent, coupled with high-performance liquid...
A bar micro-solid phase (bar μ-SPE) extraction method using either graphene or zeolite or their mixtures as an adsorbent, coupled with high-performance liquid chromatography (using a C1 column) was developed for the simultaneous determination of pharmaceutical compounds (metformin (MET), buformin (BUF), phenformin (PHEN) and propranolol (PROP)) of diverse polarity (log from -1.82 to 3.10). Parameters influencing the extraction, such as conditioning solvents, pH of the sample, sample volume, amount of adsorbent, stirring rate, time of extraction, type and volume of desorption solvent and time of desorption were investigated. Under the optimized conditions, the extraction method using graphene (extraction efficiency, % EE, ∼6-15%) resulted in the least amount of extracted drugs. However, the use of zeolite and zeolite/graphene mixtures improves the % EE significantly, 30% for PHEN and 42% for PROP using zeolite; 22% for MET and 18% for BUF using the adsorbent mixture. Under similar conditions, enrichment factors for these drugs range from 11-15. The validated method was performed for the determination of the drugs that were spiked to urine samples. Good recoveries ranging from 72.8 to 116% were achieved.
PubMed: 35479128
DOI: 10.1039/d1ra01569a -
Cancer Biology & Therapy Dec 2021Biguanide drugs (metformin and phenformin) have drawn interest for potential cancer treatments, and laboratory studies show that some cancer cells are selectively...
Biguanide drugs (metformin and phenformin) have drawn interest for potential cancer treatments, and laboratory studies show that some cancer cells are selectively sensitive to growth-inhibitory effects of biguanides. Examining metabolic pathways affected by biguanide treatments in cancer cells that are highly sensitive to biguanides, we found that biguanide treatment depletes cellular levels of both aspartate and NAD+. Experiments to replenish these metabolites or block steps of the aspartate-malate shuttle suggest that depletion of both metabolites, rather than either aspartate of NAD+ individually, is critical for growth-inhibitory effects of biguanide exposure. Even in sensitive cancer cells, though, biguanide treatment alone over a broad range of doses only inhibits cell replication without significantly affecting cell viability. Noting that clinical observations of biguanide efficacy have used combinations of agents that typically include cisplatin, we found that biguanide treatment at a cytostatic level substantially decreases survival of lung cancer and breast cancer cells when co-treated with cisplatin at doses that alone are also non-cytotoxic. This striking enhancement of cisplatin toxicity by biguanides depends on reductions of levels of NAD+ and aspartate, since addition of either of these metabolites prevented this potentiation of cisplatin cytotoxicity. Thus, biguanide drugs can have cytotoxic effects when used in combination with other cancer drugs, such as cisplatin, and depleting cellular levels of NAD+ and aspartate is critical for enhancing the cytotoxicity of cisplatin by biguanide drugs in sensitive cancer cells.
Topics: Antineoplastic Agents; Aspartic Acid; Cisplatin; Metformin; NAD; Neoplasms; Pharmaceutical Preparations
PubMed: 34720054
DOI: 10.1080/15384047.2021.1982599 -
Cancer Research Feb 2021Lung cancer is a prevalent and lethal cancer type that leads to more deaths than the next four major cancer types combined. Metastatic cancer spread is responsible for...
Lung cancer is a prevalent and lethal cancer type that leads to more deaths than the next four major cancer types combined. Metastatic cancer spread is responsible for most cancer-related deaths but the cellular changes that enable cancer cells to leave the primary tumor and establish inoperable and lethal metastases remain poorly understood. To uncover genes that are specifically required to sustain metastasis survival or growth, we performed a genome-scale pooled lentiviral-shRNA library screen in cells that represent nonmetastatic and metastatic states of lung adenocarcinoma. Mitochondrial ribosome and mitochondria-associated genes were identified as top gene sets associated with metastasis-specific lethality. Metastasis-derived cell lines and metastases analyzed from an autochthonous lung cancer mouse model had lower mitochondrial membrane potential and reduced mitochondrial functionality than nonmetastatic primary tumors. Electron microscopy of metastases uncovered irregular mitochondria with bridging and loss of normal membrane structure. Consistent with these findings, compounds that inhibit mitochondrial translation or replication had a greater effect on the growth of metastasis-derived cells. Finally, mice with established tumors developed fewer metastases upon treatment with phenformin . These results suggest that the metastatic cell state in lung adenocarcinoma is associated with a specifically altered mitochondrial functionality that can be therapeutically exploited. SIGNIFICANCE: This study characterizes altered mitochondria functionality of the metastatic cell state in lung cancer and opens new avenues for metastasis-specific therapeutic targeting.
Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Disease Models, Animal; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome-Wide Association Study; Humans; Lung Neoplasms; Mice, Knockout; Mice, Transgenic; Mitochondria; Neoplasm Metastasis; RNA Interference; Mice
PubMed: 33239425
DOI: 10.1158/0008-5472.CAN-20-1865 -
Biomedicine & Pharmacotherapy =... Aug 2020Pancreatic stellate cells (PSCs) are the main effector cells in the development of pancreatic fibrosis. Finding substances that inhibit PSC activation is an important...
Pancreatic stellate cells (PSCs) are the main effector cells in the development of pancreatic fibrosis. Finding substances that inhibit PSC activation is an important approach to inhibiting pancreatic fibrosis. Saikosaponin A (SSa) has numerous pharmacological activities, but its effect on PSCs remains unknown. This study was conducted to explore the effects of SSa on PSC activation in cultured rat PSCs. Cell viability, proliferation, migration and apoptosis were evaluated by MTT assays, the iCELLigence System, Transwell assays and flow cytometry. Markers of PSC activation, autophagy and the NLRP3 inflammasome were measured by real-time PCR, immunofluorescence and western blotting. Rapamycin and phenformin hydrochloride were used to determine the effect of SSa via the AMPK/mTOR pathway. The results showed that SSa suppressed PSC viability, proliferation, and migration and promoted apoptosis. SSa inhibited PSC activation, restrained PSC autophagy and suppressed the NLRP3 inflammasome. In addition, there was interaction between autophagy and the NLRP3 inflammasome during SSa inhibition of PSCs. Moreover, promotion of p-AMPK increased autophagy and the NLRP3 inflammasome. Inhibition of p-mTOR increased autophagy and decreased the NLRP3 inflammasome. Our results indicated that SSa inhibited PSC activation by inhibiting PSC autophagy and the NLRP3 inflammasome via the AMPK/mTOR pathway. These findings provide a theoretical basis for the use of SSa to treat pancreatic fibrosis and further suggest that targeting autophagy and the NLRP3 inflammasome may provide new strategies for the treatment of pancreatic fibrosis.
Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Autophagy; Cell Movement; Cell Proliferation; Cells, Cultured; Fibrosis; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Oleanolic Acid; Pancreatic Stellate Cells; Rats; Saponins; Signal Transduction; TOR Serine-Threonine Kinases
PubMed: 32497863
DOI: 10.1016/j.biopha.2020.110216 -
Frontiers in Pharmacology 2019Activations of Akt or ERK pathway induced by clinical drugs promote therapeutic failure due to decrease of drug response, and no available strategies have been developed...
Activations of Akt or ERK pathway induced by clinical drugs promote therapeutic failure due to decrease of drug response, and no available strategies have been developed to solve these problems. In this study, we found that pirarubicin (THP), one important chemotherapeutic drug for treating bladder cancer intravesically, dramatically elevated phosphorylations of both Akt and Erk1/2 in addition to inducing DNA damage. MK2206 or AZD6244, representative Akt and Erk1/2 inhibitors, respectively, profoundly sensitized bladder cancer cells to THP treatment. Interestingly, we found that inhibition of a single arm of either Akt or Erk1/2 pathway would induce the increase of another arm, indicating the existence of the crosstalk between these two pathways. Thus, simultaneous suppression of both signals may be needed for increasing the sensitivity of THP. On the other hand, we revealed that phenformin efficiently inhibited both Akt and Erk1/2 phosphorylation in a dose-dependent manner. Furthermore, we demonstrated that phenformin, mimicking dual inhibitors, plays dramatically synergistic action with THP both and . Our findings suggest that combination therapy of THP with dual inhibitors may constitute a successful strategy for improving chemotherapy response.
PubMed: 31649535
DOI: 10.3389/fphar.2019.01159 -
The Journal of Toxicological Sciences 2022The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the...
The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.
Topics: Animals; Cells, Cultured; Glycolysis; Hepatocytes; Liver; Mice; Oxidative Phosphorylation; Oxygen Consumption; Perfusion
PubMed: 34987137
DOI: 10.2131/jts.47.13 -
International Journal of Molecular... Aug 2021Microglial functioning depends on Ca signaling. By using Ca sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca...
Microglial functioning depends on Ca signaling. By using Ca sensitive fluorescence dye, we studied how inhibition of mitochondrial respiration changed spontaneous Ca signals in soma of microglial cells from 5-7-day-old rats grown under normoxic and mild-hypoxic conditions. In microglia under normoxic conditions, metformin or rotenone elevated the rate and the amplitude of Ca signals 10-15 min after drug application. Addition of cyclosporin A, a blocker of mitochondrial permeability transition pore (mPTP), antioxidant trolox, or inositol 1,4,5-trisphosphate receptor (IP3R) blocker caffeine in the presence of rotenone reduced the elevated rate and the amplitude of the signals implying sensitivity to reactive oxygen species (ROS), and involvement of mitochondrial mPTP together with IP3R. Microglial cells exposed to mild hypoxic conditions for 24 h showed elevated rate and increased amplitude of Ca signals. Application of metformin or rotenone but not phenformin before mild hypoxia reduced this elevated rate. Thus, metformin and rotenone had the opposing fast action in normoxia after 10-15 min and the slow action during 24 h mild-hypoxia implying activation of different signaling pathways. The slow action of metformin through inhibition of complex I could stabilize Ca homeostasis after mild hypoxia and could be important for reduction of ischemia-induced microglial activation.
Topics: Animals; Caffeine; Calcium Signaling; Chromans; Cyclosporine; Electron Transport Complex I; Female; Hypoxia; Male; Metformin; Microglia; Mitochondria; Mitochondrial Membrane Transport Proteins; Neurons; Primary Cell Culture; Rats; Reactive Oxygen Species; Rotenone
PubMed: 34502402
DOI: 10.3390/ijms22179493 -
Frontiers in Cell and Developmental... 2021Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which...
Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-Ras ) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 , as well as To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-Ras promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-Ras overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-Ras , which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.
PubMed: 34350189
DOI: 10.3389/fcell.2021.720111 -
IScience Feb 2024Metformin is the first-line treatment for type 2 diabetes, yet its mechanism of action is not fully understood. Recent studies suggest metformin's interactions with gut...
Metformin is the first-line treatment for type 2 diabetes, yet its mechanism of action is not fully understood. Recent studies suggest metformin's interactions with gut microbiota are responsible for exerting therapeutic effects. In this study, we report that metformin targets the gut microbial enzyme agmatinase, as a competitive inhibitor, which may impair gut agmatine catabolism. The metformin inhibition constant (K) of agmatinase is 1 mM and relevant in the gut where the drug concentration is 1-10 mM. Metformin analogs phenformin, buformin, and galegine are even more potent inhibitors of agmatinase (K = 0.6, 0.1, and 0.007 mM, respectively) suggesting a shared mechanism. Agmatine is a known effector of human host metabolism and has been reported to augment metformin's therapeutic effects for type 2 diabetes. This gut-derived inhibition mechanism gives new insights on metformin's action in the gut and may lead to significant discoveries in improving metformin therapy.
PubMed: 38318350
DOI: 10.1016/j.isci.2024.108900