Unlocking a New Path: An Autophagometer that Measures Flux Using a Non-Fluorescent Immunohistochemistry Method.

Macroautophagy/autophagy, a crucial cellular process, is typically measured using fluorescence-based techniques, which can be costly, complex, and impractical for clinical settings. In this paper, we introduce a novel, cost-effective, non-fluorescent immunohistochemistry (IHC) method for evaluating autophagy flux. This technique, based on antigen-antibody reactions and chromogenic detection, provides clear, quantifiable results under standard light microscopy, eliminating the need for expensive equipment and specialized reagents. Our method simplifies technical requirements, making it accessible to routine clinical laboratories and research settings with limited resources. By comparing our approach with traditional fluorescence methods, we demonstrate its superior effectiveness, cost-efficiency, and applicability to patient samples. This innovative technique has the potential to significantly advance autophagy research and improve clinical diagnostics, offering a practical and robust tool for studying autophagy mechanisms in diseases such as cancer and neurodegenerative disorders. Our non-fluorescent IHC method represents a significant step forward in evaluating autophagy flux, making it more accessible and reliable, with the promise of enhancing our understanding and treatment of autophagy-related diseases.

Mechanistic insights into mesenchymal-amoeboid transition as an intelligent cellular adaptation in cancer metastasis and resistance.

Malignant cell plasticity is an important hallmark of tumor biology and crucial for metastasis and resistance. Cell plasticity lets cancer cells adapt to and escape the therapeutic strategies, which is the leading cause of cancer patient mortality. Epithelial cells acquire mobility via epithelial-mesenchymal transition (EMT), whereas mesenchymal cells enhance their migratory ability and clonogenic potential by acquiring amoeboid characteristics through mesenchymal-amoeboid transition (MAT). Tumor formation, progression, and metastasis depend on the tumor microenvironment (TME), a complex ecosystem within and around a tumor. Through increased migration and metastasis of cancer cells, the TME also contributes to malignancy. This review underscores the distinction between invasion pattern morphological manifestations and the diverse structures found within the TME. Furthermore, the mechanisms by which amoeboid-associated characteristics promote resistance and metastasis and how these mechanisms may represent therapeutic opportunities are discussed.

CSF amino acid profiles in ICV-streptozotocin-induced sporadic Alzheimer's disease in male Wistar rat: a metabolomics and systems biology perspective.

Alzheimer's disease (AD) is an increasingly important public health concern due to the increasing proportion of older individuals within the general population. The impairment of processes responsible for adequate brain energy supply primarily determines the early features of the aging process. Restricting brain energy supply results in brain hypometabolism prior to clinical symptoms and is anatomically and functionally associated with cognitive impairment. The present study investigated changes in metabolic profiles induced by intracerebroventricular-streptozotocin (ICV-STZ) in an AD-like animal model. To this end, male Wistar rats received a single injection of STZ (3 mg·kg-1) by ICV (2.5 µL into each ventricle for 5 min on each side). In the second week after receiving ICV-STZ, rats were tested for cognitive performance using the Morris Water Maze test and subsequently prepared for positron emission tomography (PET) to confirm AD-like symptoms. Tandem Mass Spectrometry (MS/MS) analysis was used to detect amino acid changes in cerebrospinal fluid (CFS) samples. Our metabolomics study revealed a reduction in the concentrations of various amino acids (alanine, arginine, aspartic acid, glutamic acid, glycine, isoleucine, methionine, phenylalanine, proline, serine, threonine, tryptophane, tyrosine, and valine) in CSF of ICV-STZ-treated animals as compared to controls rats. The results of the current study indicate amino acid levels could potentially be considered targets of nutritional and/or pharmacological interventions to interfere with AD progression.

Correction: Dastghaib et al. Simvastatin Induces Unfolded Protein Response and Enhances Temozolomide-Induced Cell Death in Glioblastoma Cells. Cells 2020, 9, 2339.

In the original publication [...].

Autophagy and Biomaterials: A Brief Overview of the Impact of Autophagy in Biomaterial Applications.

Macroautophagy (hereafter autophagy), a tightly regulated physiological process that obliterates dysfunctional and damaged organelles and proteins, has a crucial role when biomaterials are applied for various purposes, including diagnosis, treatment, tissue engineering, and targeted drug delivery. The unparalleled physiochemical properties of nanomaterials make them a key component of medical strategies in different areas, such as osteogenesis, angiogenesis, neurodegenerative disease treatment, and cancer therapy. The application of implants and their modulatory effects on autophagy have been known in recent years. However, more studies are necessary to clarify the interactions and all the involved mechanisms. The advantages and disadvantages of nanomaterial-mediated autophagy need serious attention in both the biological and bioengineering fields. In this mini-review, the role of autophagy after biomaterial exploitation and the possible related mechanisms are explored.

Ceramides and ceramide synthases in cancer: Focus on apoptosis and autophagy.

Different studies corroborate a role for ceramide synthases and their downstream products, ceramides, in modulation of apoptosis and autophagy in the context of cancer. These mechanisms of regulation, however, appear to be context dependent in terms of ceramides' fatty acid chain length, subcellular localization, and the presence or absence of their downstream targets. Our current understanding of the role of ceramide synthases and ceramides in regulation of apoptosis and autophagy could be harnessed to pioneer the development of new treatments to activate or inhibit a single type of ceramide synthase, thereby regulating the apoptosis induction or cross talk of apoptosis and autophagy in cancer cells. Moreover, the apoptotic function of ceramide suggests that ceramide analogues can pave the way for the development of novel cancer treatments. Therefore, in the current review paper we discuss the impact of ceramide synthases and ceramides in regulation of apoptosis and autophagy in context of different types of cancers. We also briefly introduce the latest information on ceramide synthase inhibitors, their application in diseases including cancer therapy, and discuss approaches for drug discovery in the field of ceramide synthase inhibitors. We finally discussed strategies for developing strategies to use lipids and ceramides analysis in biological fluids for developing early biomarkers for cancer.

Suppression of academics and school training in Iran during the "Woman, Life, Freedom" revolution.

For the past 6 months, there has been an ongoing revolution in Iran after the brutal death of Zhina (Mahsa) Amini in morality police custody. Iranian universities' professors and students have been on the frontline of this revolution and have been fired or sentenced. On the other hand, Iranian high schools and primary schools have been under suspected toxic gas attack. In the current article, the latest status of oppression of the university students and professors and toxic gas attack on primary and high schools in Iran has been evaluated.

Placental Nanoparticle Uptake-On-a-Chip: The Impact of Trophoblast Syncytialization and Shear Stress.

The placenta is a dynamic and complex organ that plays an essential role in the health and development of the fetus. Placental disorders can affect the health of both the mother and the fetus. There is currently an unmet clinical need to develop nanoparticle-based therapies to target and treat placental disorders. However, little is known about the interaction of nanoparticles (NPs) with the human placenta under biomimetic conditions. Specifically, the impact of shear stress exerted on the trophoblasts (placental epithelial cells) by the maternal blood flow, the gradual fusion of the trophoblasts along the gestation period (syncytialization), and the impact of microvilli formation on the cell uptake of NPs is not known. To this end, we designed dynamic placenta-on-a-chip models using BeWo cells to recapitulate the micro-physiological environment, and we induced different degrees of syncytialization via chemical induction with forskolin. We characterized the degree of syncytialization quantitatively by measuring beta human chorionic gonadotropin (ß-hCG) secretion, as well as qualitatively by immunostaining the tight junction protein, ZO-1, and counter nuclear staining. We also characterized microvilli formation under static and dynamic conditions via F-actin staining. We used these models to measure the cell uptake of chondroitin sulfate a binding protein (CSA) conjugated and control liposomes using confocal microscopy, followed by image analysis. Interestingly, exposure of the cells to a dynamic flow of media intrinsically induced syncytialization and microvilli formation compared to static controls. Under dynamic conditions, BeWo cells produced more ß-hCG in conditions that increased the cell exposure time to forskolin (p < 0.005). Our cell uptake results clearly show a combined effect of the exerted shear stress and forskolin treatment on the cell uptake of liposomes as uptake increased in forskolin exposed conditions (p < 0.05). Overall, the difference in the extent of cell uptake of liposomes among the different conditions clearly displays a need for the development of dynamic models of the placenta that consider the changes in the placental cell phenotype along the gestation period, including syncytialization, microvilli formation, and the expression of different transport and uptake receptors. Knowledge generated from this work will inform future research aiming at developing drug delivery systems targeting the placenta.

Multifunctional Thermoresponsive Microcarriers for High-Throughput Cell Culture and Enzyme-Free Cell Harvesting.

An effective treatment of human diseases using regenerative medicine and cell therapy approaches requires a large number of cells. Cultivation of cells on microcarriers is a promising approach due to the high surface-to-volume ratios that these microcarriers offer. Here, multifunctional temperature-responsive microcarriers (cytoGel) made of an interpenetrating hydrogel network composed of poly(N-isopropylacrylamide) (PNIPAM), poly(ethylene glycol) diacrylate (PEGDA), and gelatin methacryloyl (GelMA) are developed. A flow-focusing microfluidic chip is used to produce microcarriers with diameters in the range of 100-300 µm and uniform size distribution (polydispersity index of ≈0.08). The mechanical properties and cells adhesion properties of cytoGel are adjusted by changing the composition hydrogel composition. Notably, GelMA regulates the temperature response and enhances microcarrier stiffness. Human-derived glioma cells (U87) are grown on cytoGel in static and dynamic culture conditions with cell viabilities greater than 90%. Enzyme-free cell detachment is achieved at room temperature with up to 70% detachment efficiency. Controlled release of bioactive molecules from cytoGel is accomplished for over a week to showcase the potential use of microcarriers for localized delivery of growth factors to cell surfaces. These microcarriers hold great promise for the efficient expansion of cells for the industrial-scale culture of therapeutic cells.

Vitamin D3 and estradiol alter PAD2 expression and activity levels in C6 glioma cells.

Multiple Sclerosis (MS) is known as a chronic demyelinating disease with multifactorial etiology. It is suggested that the deimination of myelin basic proteins (MBPs) by peptidyl arginine deiminase 2 (PAD2) may increase citrulline residues resulting in the reduction of myelin sheath density and the progression of multiple sclerosis. The aim of this study was to investigate the effects of vitamin D (25-hydroxy cholecalciferol (D3)) and estradiol on PAD2 gene expression level and its catalytic activity in rat C6 glioma cells. C6 glioma cells were cultured in DMEM medium and were treated with vitamin D (10 and 100 ng/ml) and estradiol (10 and 100 µM) based on the cellular viability. Then, the PAD2 gene expression and catalytic activity were evaluated using real-time qRT-PCR and spectrophotometry techniques, respectively. The PAD2 gene expression level and its catalytic activity increased significantly in estradiol-treated cells (P = 0.0435 and P = 0.0015, respectively). Conversely, vitamin D downregulated significantly the PAD2 gene expression level (P < 0.015) and its activity (P < 0.017). The study results suggested that estradiol conversely with vitamin D increases the activity of the PAD2 enzyme so that it might develop multiple sclerosis, especially in women.

Dynamic placenta-on-a-chip model for fetal risk assessment of nanoparticles intended to treat pregnancy-associated diseases.

Pregnant women often have to take medication either for pregnancy-related diseases or for previously existing medical conditions. Current maternal medications pose fetal risks due to off target accumulation in the fetus. Nanoparticles, engineered particles in the nanometer scale, have been used for targeted drug delivery to the site of action without off-target effects. This has opened new avenues for treatment of pregnancy-associated diseases while minimizing risks on the fetus. It is therefore instrumental to study the potential transfer of nanoparticles from the mother to the fetus. Due to limitations of in vivo and ex vivo models, an in vitro model mimicking the in vivo situation is essential. Placenta-on-a-chip provides a microphysiological recapitulation of the human placenta. Here, we reviewed the fetal risks associated with current therapeutic approaches during pregnancy, analyzed the advantages and limitations of current models used for nanoparticle assessment, and highlighted the current need for using dynamic placenta-on-a-chip models for assessing the safety of novel nanoparticle-based therapies during pregnancy.

Statins in patients with COVID-19: a retrospective cohort study in Iranian COVID-19 patients.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has profoundly affected the lives of millions of people. To date, there is no approved vaccine or specific drug to prevent or treat COVID-19, while the infection is globally spreading at an alarming rate. Because the development of effective vaccines or novel drugs could take several months (if not years), repurposing existing drugs is considered a more efficient strategy that could save lives now. Statins constitute a class of lipid-lowering drugs with proven safety profiles and various known beneficial pleiotropic effects. Our previous investigations showed that statins have antiviral effects and are involved in the process of wound healing in the lung. This triggered us to evaluate if statin use reduces mortality in COVID-19 patients. RESULTS: After initial recruitment of 459 patients with COVID-19 (Shiraz province, Iran) and careful consideration of the exclusion criteria, a total of 150 patients, of which 75 received statins, were included in our retrospective study. Cox proportional-hazards regression models were used to estimate the association between statin use and rate of death. After propensity score matching, we found that statin use appeared to be associated with a lower risk of morbidity [HR = 0.85, 95% CI = (0.02, 3.93), P = 0.762] and lower risk of death [(HR = 0.76; 95% CI = (0.16, 3.72), P = 0.735)]; however, these associations did not reach statistical significance. Furthermore, statin use reduced the chance of being subjected to mechanical ventilation [OR = 0.96, 95% CI = (0.61-2.99), P = 0.942] and patients on statins showed a more normal computed tomography (CT) scan result [OR = 0.41, 95% CI = (0.07-2.33), P = 0.312]. CONCLUSIONS: Although we could not demonstrate a significant association between statin use and a reduction in mortality in patients with COVID19, we do feel that our results are promising and of clinical relevance and warrant the need for prospective randomized controlled trials and extensive retrospective studies to further evaluate and validate the potential beneficial effects of statin treatment on clinical symptoms and mortality rates associated with COVID-19.

Simvastatin Induces Unfolded Protein Response and Enhances Temozolomide-Induced Cell Death in Glioblastoma Cells.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor with a very poor survival rate. Temozolomide (TMZ) is the common chemotherapeutic agent used for GBM treatment. We recently demonstrated that simvastatin (Simva) increases TMZ-induced apoptosis via the inhibition of autophagic flux in GBM cells. Considering the role of the unfolded protein response (UPR) pathway in the regulation of autophagy, we investigated the involvement of UPR in Simva-TMZ-induced cell death by utilizing highly selective IRE1 RNase activity inhibitor MKC8866, PERK inhibitor GSK-2606414 (PERKi), and eIF2α inhibitor salubrinal. Simva-TMZ treatment decreased the viability of GBM cells and significantly increased apoptotic cell death when compared to TMZ or Simva alone. Simva-TMZ induced both UPR, as determined by an increase in GRP78, XBP splicing, eukaryote initiation factor 2α (eIF2α) phosphorylation, and inhibited autophagic flux (accumulation of LC3ß-II and inhibition of p62 degradation). IRE1 RNase inhibition did not affect Simva-TMZ-induced cell death, but it significantly induced p62 degradation and increased the microtubule-associated proteins light chain 3 (LC3)ß-II/LC3ß-I ratio in U87 cells, while salubrinal did not affect the Simva-TMZ induced cytotoxicity of GBM cells. In contrast, protein kinase RNA-like endoplasmic reticulum kinase (PERK) inhibition significantly increased Simva-TMZ-induced cell death in U87 cells. Interestingly, whereas PERK inhibition induced p62 accumulation in both GBM cell lines, it differentially affected the LC3ß-II/LC3ß-I ratio in U87 (decrease) and U251 (increase) cells. Simvastatin sensitizes GBM cells to TMZ-induced cell death via a mechanism that involves autophagy and UPR pathways. More specifically, our results imply that the IRE1 and PERK signaling arms of the UPR regulate Simva-TMZ-mediated autophagy flux inhibition in U251 and U87 GBM cells.

Pleiotropic effects of statins: A focus on cancer.

The statin drugs ('statins') potently inhibit hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase by competitively blocking the active site of the enzyme. Statins decrease de novo cholesterol biosynthesis and thereby reduce plasma cholesterol levels. Statins exhibit "pleiotropic" properties that are independent of their lipid-lowering effects. For example, preclinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. Furthermore, statins show chemo-sensitizing effects by impairing Ras family GTPase signaling. However, whether statins have clinically meaningful anti-cancer effects remains an area of active investigation. Both preclinical and clinical studies on the potential mechanisms of action of statins in several cancers have been reviewed in the literature. Considering the contradictory data on their efficacy, we present an up-to-date summary of the pleiotropic effects of statins in cancer therapy and review their impact on different malignancies. We also discuss the synergistic anti-cancer effects of statins when combined with other more conventional anti-cancer drugs to highlight areas of potential therapeutic development.

Autophagy and SARS-CoV-2 infection: Apossible smart targeting of the autophagy pathway.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak resulted in 5,993,317 confirmed cases worldwide with 365,394 confirmed deaths (as of May 29th, 2020, WHO). The molecular mechanism of virus infection and spread in the body is not yet disclosed, but studies on other betacoronaviruses show that, upon cell infection, these viruses inhibit macroautophagy/autophagy flux and cause the accumulation of autophagosomes. No drug has yet been approved for the treatment of SARS-CoV-2 infection; however, preclinical investigations suggested repurposing of several FDA-approved drugs for clinical trials. Half of these drugs are modulators of the autophagy pathway. Unexpectedly, instead of acting by directly antagonizing the effects of viruses, these drugs appear to function by suppressing autophagy flux. Based on the established cross-talk between autophagy and apoptosis, we speculate that over-accumulation of autophagosomes activates an apoptotic pathway that results in apoptotic death of the infected cells and disrupts the virus replication cycle. However, administration of the suggested drugs are associated with severe adverse effects due to their off-target accumulation. Nanoparticle targeting of autophagy at the sites of interest could be a powerful tool to efficiently overcome SARS-CoV-2 infection while avoiding the common adverse effects of these drugs.

Prevalence and Associated Factors of Anxiety and Depression Among Patients with Type 2 Diabetes in Kerman, Southern Iran.

PURPOSE: Depression and anxiety are common disorders in patients suffering from type 2 diabetes. These disorders can lead to premature morbidity, exacerbate disease complications, make patients suffer more, and increase health-care costs. As diabetes has increased worldwide recently, it is necessary to reduce the prevalence of factors that are associated with depression and anxiety in diabetes patients. This study aimed to assess the prevalence of anxiety and depression and to identify their associated factors, including metabolic components among people with type 2 diabetes. PATIENTS AND METHODS: We performed a cross-sectional study in 1500 patients with type 2 diabetes in Kerman, in the southern part of Iran. The prevalence of depression and anxiety was estimated using the Beck Depression Inventory and the Hamilton Anxiety questionnaires, respectively. After calculating the proportions of depression and anxiety, univariate logistic regression was performed. Factors whose P-values were smaller than 0.2 in univariate logistic regression were included in multiple logistic regression for confounder adjustments. The analysis was performed using SPSS version 20. RESULTS: The rates of depression and anxiety were 59% (95% CI: 54.48-63.12) and 62% (95% CI: 59.51-66.27), respectively. Factors found to be independently associated with anxiety were high FBS, high LDL-C, high TG, hypertension, complications, low physical activity. Factors found to be independently associated with depression were female gender, older age, high BMI, high FBS, high LDL-C, low HDL-C, high TG, high HbA1c, hypertension, and low physical activity. Complications were independently associated with anxiety but not with depression. Female gender, older age, high BMI, low HDL-C, and high HbA1c were independently associated with depression but not with anxiety. CONCLUSION: Current findings demonstrated that a large proportion of patients with type 2 diabetes suffer from depression and anxiety. This study also identified factors associated with these disorders. Controlling some metabolic variables will decrease the prevalence of these disorders and improves clinical remedy and quality of life in patients with type 2 diabetes.

Facile Method for Fabrication of Meter-Long Multifunctional Hydrogel Fibers with Controllable Biophysical and Biochemical Features.

Hydrogel structures with microscale morphological features have extensive application in tissue engineering owing to their capacity to induce desired cellular behavior. Herein, we describe a novel biofabrication method for fabrication of grooved solid and hollow hydrogel fibers with control over their cross-sectional shape, surface morphology, porosity, and material composition. These fibers were further configured into three-dimensional structures using textile technologies such as weaving, braiding, and embroidering methods. Additionally, the capacity of these fibers to integrate various biochemical and biophysical cues was shown via incorporating drug-loaded microspheres, conductive materials, and magnetic particles, extending their application to smart drug delivery, wearable or implantable medical devices, and soft robotics. The efficacy of the grooved fibers to induce cellular alignment was evaluated on various cell types including myoblasts, cardiomyocytes, cardiac fibroblasts, and glioma cells. In particular, these fibers were shown to induce controlled myogenic differentiation and morphological changes, depending on their groove size, in C2C12 myoblasts.

Simvastatin increases temozolomide-induced cell death by targeting the fusion of autophagosomes and lysosomes.

Temozolomide (TMZ) is a chemotherapy agent used to treat Grade IV astrocytoma, also known as glioblastoma (GBM). TMZ treatment causes DNA damage that results in tumor cell apoptosis and increases the survival rate of GBM patients. However, chemoresistance as a result of TMZ-induced autophagy significantly reduces this anticancer effects over time. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate (MEV) cascade. Statins are best known for their cholesterol (CH)-lowering effect. Long-term consumption of statins, prior to and in parallel with other cancer therapeutic approaches, has been reported to increase the survival rate of patients with various forms of cancers. In this study, we investigated the potentiation of TMZ-induced apoptosis by simvastatin (Simva) in human GBM cell lines and patient GBM cells, using cell monolayers and three-dimensional cell culture systems. The incubation of cells with a combination of Simva and TMZ resulted in a significant increase in apoptotic cells compared to cells treated with TMZ alone. Incubation of cells with CH or MEV cascade intermediates failed to compensate the decrease in cell viability induced by the combined Simva and TMZ treatment. Simva treatment inhibited the autophagy flux induced by TMZ by blocking autophago-lysosome formation. Our results suggest that Simva sensitizes GBM cells to TMZ-induced cell death in a MEV cascade-independent manner and identifies the inhibition of autophagosome-lysosome fusion as a promising therapeutic strategy in the treatment of GBM.

The BDNF Protein and its Cognate mRNAs in the Rat Spinal Cord during Amylin-induced Reversal of Morphine Tolerance.

The pancreatic peptide, Amylin (AMY), reportedly affects nociception in rodents. Here, we investigated the potential effect of AMY on the tolerance to morphine and on the expression of BDNF at both levels of protein and RNA in the lumbar spinal cord of morphine tolerant rats. Animals in both groups of control and test received a single daily dose of intrathecal (i.t.) morphine for 10 days. Rats in the test group received AMY (1, 10 and 60 pmoles) in addition to morphine from days 6 to10. Morphine tolerance was established at day 5. AMY alone showed enduring antinociceptive effects for 10 days. Real-Time PCR, western blotting and ELISA were used respectively to assess levels of BDNF transcripts and their encoded proteins. Rats tolerant to i.t. morphine showed increased expression of exons I, IV, and IX of the BDNF gene, and had elevated levels of pro-BDNF and BDNF protein in their lumbar spinal cord. AMY, when co-administered with morphine from days 6 to 10, reversed morphine tolerance and adversely affected the morphine-induced expression of the BDNF gene at both levels of protein and mRNAs containing exons I, IV and IX. AMY alone increased levels of exons I and IV transcripts. Levels of pro-BDNF and BDNF proteins remained unchanged in the lumbar spinal cord of rats treated by AMY alone. These results suggest that i.t. AMY not only abolished morphine tolerance, but also reduced the morphine induced increase in the expression of both BDNF transcripts and protein in the lumbar spinal cord.

Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.