Construction of a screening system for lipid-derived radical inhibitors and validation of hit compounds to target retinal and cerebrovascular diseases.

Recent studies have highlighted the indispensable role of oxidized lipids in inflammatory responses, cell death, and disease pathogenesis. Consequently, inhibitors targeting oxidized lipids, particularly lipid-derived radicals critical in lipid peroxidation, which are known as radical-trapping antioxidants (RTAs), have been actively pursued. We focused our investigation on nitroxide compounds that have rapid second-order reaction rate constants for reaction with lipid-derived radicals. A novel screening system was developed by employing competitive reactions between library compounds and a newly developed profluorescence nitroxide probe with lipid-derived radicals to identify RTA compounds. A PubMed search of the top hit compounds revealed their wide application as repositioned drugs. Notably, the inhibitory efficacy of methyldopa, selected from these compounds, against retinal damage and bilateral common carotid artery stenosis was confirmed in animal models. These findings underscore the efficacy of our screening system and suggest that it is an effective approach for the discovery of RTA compounds.

Inhibition of G protein-coupled receptor 68 using homoharringtonine attenuates chronic kidney disease-associated cardiac impairment.

Chronic kidney disease (CKD) induces cardiac inflammation and fibrosis and reduces survival. We previously demonstrated that G protein-coupled receptor 68 (GPR68) promotes cardiac inflammation and fibrosis in mice with 5/6 nephrectomy (5/6Nx) and patients with CKD. However, no method of GPR68 inhibition has been found that has potential for therapeutic application. Here, we report that Cephalotaxus harringtonia var. nana extract and homoharringtonine ameliorate cardiac inflammation and fibrosis under CKD by suppressing GPR68 function. Reagents that inhibit the function of GPR68 were explored by high-throughput screening using a medicinal plant extract library (8,008 species), and we identified an extract from Cephalotaxus harringtonia var. nana as a GPR68 inhibitor that suppresses inflammatory cytokine production in a GPR68 expression-dependent manner. Consumption of the extract inhibited inflammatory cytokine expression and cardiac fibrosis and improved the decreased survival attributable to 5/6Nx. Additionally, homoharringtonine, a cephalotaxane compound characteristic of C. harringtonia, inhibited inflammatory cytokine production. Homoharringtonine administration in drinking water alleviated cardiac fibrosis and improved heart failure and survival in 5/6Nx mice. A previously unknown effect of C. harringtonia extract and homoharringtonine was revealed in which GPR68-dependent inflammation and cardiac dysfunction were suppressed. Utilizing these compounds could represent a new strategy for treating GPR68-associated diseases, including CKD.

Innovative Combination Treatment to Expand the Indications of Particle Therapy: Spacer Placement Surgery Using Bio-Absorbable Polyglycolic Acid Spacer.

BACKGROUND: Particle therapy has favorable dose distribution and high curability. However, radiotherapy for malignant tumors adjacent to the gastrointestinal tract is contraindicated owing to its low tolerance. To overcome this, combination treatment with surgery to make a space between the tumor and adjacent gastrointestinal tract followed by particle therapy has been developed. Several materials have been used for the spacer and recently, we developed the absorbable polyglycolic acid (PGA) spacer, which has been used since 2019. This study is the first report of consecutive case series of spacer placement surgery using the PGA spacer. STUDY DESIGN: Fifty consecutive patients undergoing spacer placement surgery with the PGA spacer were evaluated. Postoperative laboratory data, morbidity related to the treatment, and spacer volume after treatment were evaluated. RESULTS: There were no treatment-related deaths, and all but 2 patients completed combination treatment. The median ratios of postoperative PGA spacer volume to the pretreatment volume were 96.9%, 87.7%, and 74.6% at weeks 2, 4, and 8, respectively. The spacer volume was maintained at 80% at 7 weeks and was predicted to be 50% at 15 weeks and 20% in 24 weeks. CONCLUSIONS: Spacer placement surgery using the PGA spacer was feasible and tolerable. The PGA spacers maintained sufficient thickness during the duration of subsequent particle therapy. Combination treatment using the PGA spacer is innovative and has the potential to become a new standard curative local treatment.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

Simultaneous imaging of luminescence and prompt x-rays during irradiation with spot-scanning proton beams at clinical dose level.

Prompt x-ray imaging is a promising method for observing the beam shape from outside a subject. However, its distribution is different from dose distribution, and thus a comparison with the dose is required. Meanwhile, luminescence imaging of water is a possible method for imaging the dose distribution. Consequently, we performed simultaneous imaging of luminescence and prompt x-rays during irradiation by proton beams to compare the distributions between these two different imaging methods. Optical imaging of water was conducted with spot-scanning proton beams at clinical dose level during irradiation to a fluorescein (FS) water phantom set in a black box. Prompt x-ray imaging was also conducted simultaneously from outside the black box using a developed x-ray camera during proton beam irradiation to the phantom. We measured images of the luminescence of FS water and prompt x-rays for various types of proton beams, including pencil beams, spread-out Bragg peak (SOBP) beams, and clinically used therapy beams. After the imaging, ranges were estimated from FS water and prompt x-rays and compared with those calculated with a treatment planning system (TPS). We could measure the prompt x-ray and FS water images simultaneously for all types of proton beams. The ranges estimated from the FS water and those calculated with the TPS closely matched, within a difference of several mm. Similar range difference was found between the results estimated from prompt x-ray images and those calculated with the TPS. We confirmed that the simultaneous imaging of luminescence and prompt x-rays were possible during irradiation with spot-scanning proton beams at a clinical dose level. This method can be applied to range estimation as well as comparison with the dose for prompt x-ray imaging or other imaging methods used in therapy with various types of proton beams at a clinical dose level.

Formation of spread-out Bragg peak for helium-ion beam using microdosimetric kinetic model.

PURPOSE: To evaluate the applicability of microdosimetric kinetic model (MKM) to helium-ion therapy by forming a spread-out Bragg peak (SOBP) of a helium-ion beam using the MKM developed for carbon-ion radiotherapy and confirming the predictions in biological experiments. METHODS: Using a ridge filter, a 90-mm wide SOBP for a 210 MeV/u helium-ion beam was created in a broad beam delivery system. The ridge filter was designed such that a uniform biological response was achieved with a cell survival rate of 7% over the SOBP region. Biological experiments were then performed using the SOBP beam in a human salivary gland (HSG) cell line to measure the cell survival rates. RESULTS: The biological responses were uniform in the SOBP region, as expected by the MKM; however, the mean of the measured cell survival rates was (11.2 ± 0.6) % in the SOBP region, which was 60% higher than the designed rate. When investigating the biological parameters of the HSG cell line used in the experiments, we found that they were altered slightly from the MKM parameters used for carbon-ion radiotherapy. The new ß parameter reproduced the measured survival rates within 6.5% in the SOBP region. CONCLUSION: We produced biologically uniform SOBP using MKM for carbon-ion radiotherapy. The measured survival rates in the SOBP region were higher than expected, and the survival rates were reproduced by modifying the MKM parameter. This study was limited to one SOBP, and further investigations are required to prove that MKM is generally applicable to helium-ion radiotherapy.

GPR55 contributes to neutrophil recruitment and mechanical pain induction after spinal cord compression in mice.

Pain transmission and processing in the nervous system are modulated by various biologically active substances, including lysophospholipids, through direct and indirect actions on the somatosensory pathway. Lysophosphatidylglucoside (LysoPtdGlc) was recently identified as a structurally unique lysophospholipid that exerts biological actions via the G protein-coupled receptor GPR55. Here, we demonstrated that GPR55-knockout (KO) mice show impaired induction of mechanical pain hypersensitivity in a model of spinal cord compression (SCC) without the same change in the models of peripheral tissue inflammation and peripheral nerve injury. Among these models, only SCC recruited peripheral inflammatory cells (neutrophils, monocytes/macrophages, and CD3+ T-cells) in the spinal dorsal horn (SDH), and GPR55-KO blunted these recruitments. Neutrophils were the first cells recruited to the SDH, and their depletion suppressed the induction of SCC-induced mechanical hypersensitivity and inflammatory responses in compressed SDH. Furthermore, we found that PtdGlc was present in the SDH and that intrathecal administration of an inhibitor of secretory phospholipase A2 (an enzyme required for producing LysoPtdGlc from PtdGlc) reduced neutrophil recruitment to compressed SDH and suppressed pain induction. Finally, by screening compounds from a chemical library, we identified auranofin as a clinically used drug with an inhibitory effect on mouse and human GPR55. Systemically administered auranofin to mice with SCC effectively suppressed spinal neutrophil infiltration and pain hypersensitivity. These results suggest that GPR55 signaling contributes to the induction of inflammatory responses and chronic pain after SCC via the recruitment of neutrophils and may provide a new target for reducing pain induction after spinal cord compression, such as spinal canal stenosis.

Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

Fine-Tuning of Piezo1 Expression and Activity Ensures Efficient Myoblast Fusion during Skeletal Myogenesis.

Mechanical stimuli, such as stretch and resistance training, are essential in regulating the growth and functioning of skeletal muscles. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigated the role of the mechanosensitive ion channel Piezo1 during myogenic progression of both fast and slow muscle satellite cells. We found that Piezo1 level increases during myogenic differentiation and direct manipulation of Piezo1 in muscle stem cells alters the myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion, leading to smaller myotubes. Such an event is accompanied by significant downregulation of the fusogenic protein Myomaker. In parallel, while Piezo1 knockdown also lowers Ca2+ influx in response to stretch, Piezo1 activation increases Ca2+ influx in response to stretch and enhances myoblasts fusion. These findings may help understand molecular defects present in some muscle diseases. Our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases, including muscular dystrophies and neuromuscular disorders.

Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

Simulation study using the spots deletion technique in spot scanning proton beam therapy for prostate cancers.

The aim of the present study was to investigate the effects on the dose distribution and beam delivery time in spot scanning proton beam therapy (PBT) incorporating the spot deletion technique. A spot scanning plan was created for 30 patients with prostate cancer. The plan was then modified via two processes: Spots with lower weighting depositions were deleted (process A) and spots that were distant from the clinical target volume (CTV) were deleted (process B). The dose distribution to the organs at risk (OAR), the expanded CTV (exCTV), which was defined by a uniform expansion of the CTV by a radius of 5 mm, and the beam delivery time were compared among initial and modified plans. The V50 Gy [relative biological effectiveness (RBE)] to the rectum and bladder, and V60 Gy(RBE) to the urethral bulb, inhomogeneity index (INH) of the exCTV showed a difference (P=1.1x10-14, P=6.4x10-14, P=2.7x10-7, P=3.2x10-17), although only changes by process B were significant. Modified plan by process B showed the V50 Gy(RBE) to the rectum and bladder decreased by -2.4±1.6 and -2.3±1.4%, and the V60 Gy (RBE) to the urethral bulb decreased by -15.9±19.4%. The INH of the exCTV increased by 0.05±0.03%. On the other hand, modification of the initial plan by process A did not affect the dose of the OAR, exCTV or beam delivery time. In spot scanning PBT, modification of the initial radiotherapy plan by systemic deletion of spots distant from the CTV could result in a dose reduction to the OAR.

Dose distribution effects of spot-scanning proton beam therapy equipped with a multi-leaf collimator for pediatric brain tumors.

The present study simulated the effect of spot-scanning proton beam therapy (PBT) performed using a device equipped with a multi-leaf collimator (MLC) to calculate the dose distribution. Simulation studies using 18 pediatric patients with brain tumors in the posterior fossa were performed. Treatment plans were created for the MLC at different stages: Fully open (initial plan), fully closed to allow an irradiated area extending to 15 mm from the clinical target volume (CTV) (15-mm plan), or closing only the leaves where an organ at risk (OAR) overlapped with a border at 10 or 5 mm from the CTV (10- and 5-mm plans, respectively). The mean dose values for the brainstem, cervical cord, brain and cochlea in all MLC closure plans decreased as the MLC was closed (P=9.9×10-10, P=1.3×10-17, P=2.1×10-16 and P=2.0×10-5, respectively). The maximum dose (Dmax) values of the cervical cord and cochlea in all MLC closure plans were also decreased as the MLC was closed (P=3.0×10-4 and P=1.1×10-5, respectively). The dose to the CTV was almost unchanged. In 10 patients, the Dmax of the brain in all MLC-closure plans was higher than that of the initial plan, but the maximum increase was only 0.8 gray relative biological effectiveness [Gy(RBE)]. In conclusion, the existing MLC installed in the treatment device can be used to decrease the OAR dose significantly using spot-scanning PBT without a large capital investment. The dose from the scattered particles was small.

New high-throughput screening detects compounds that suppress pancreatic stellate cell activation and attenuate pancreatic cancer growth.

BACKGROUND/OBJECTIVES: Pancreatic stellate cells (PSCs) are involved in abundant desmoplasia, which promotes cancer cell aggressiveness and resistance to anti-cancer drugs. Therefore, PSCs are suggested to be a promising therapeutic target by attenuating PSC activation to inhibit tumor-stromal interactions with pancreatic cancer cells. Here, we developed a screen to identify compounds that reduce the activity of PSCs and investigated the effect of candidates on pancreatic cancer. METHODS: Lipid droplet accumulation in PSCs was used to observe differences in PSC activity and a new high-throughput screening platform that quantified lipid droplets in PSCs was established. A library of 3398 Food and Drug Administration-approved drugs was screened by this platform. Validation assays were performed in vitro and in vivo. RESULTS: Thirty-two compounds were finally selected as candidate compounds by screening. These compounds decreased α-smooth muscle actin expression and inhibited autophagic flux in PSCs in vitro. Among the candidates, three drugs selected for validation assays inhibited the proliferation and migration of PSCs and invasion of cancer cells by disrupting tumor-stromal interactions. Production of extracellular matrix molecules was also decreased significantly by this treatment. In vivo testing in xenograft models showed that dopamine antagonist zuclopenthixol suppressed tumor growth; this suppression was significantly increased when combined with gemcitabine. CONCLUSIONS: A new screening platform that focused on the morphological features of PSCs was developed. Candidate drugs from this screening suppressed PSC activation and tumor growth. This screening system may be useful to discover new compounds that attenuate PSC activation.

New Inhibitory Effects of Cilnidipine on Microglial P2X7 Receptors and IL-1ß Release: An Involvement in its Alleviating Effect on Neuropathic Pain.

P2X7 receptors (P2X7Rs) belong to a family of ATP-gated non-selective cation channels. Microglia represent a major cell type expressing P2X7Rs. The activation of microglial P2X7Rs causes the release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß). This response has been implicated in neuroinflammatory states in the central nervous system and in various diseases, including neuropathic pain. Thus, P2X7R may represent a potential therapeutic target. In the present study, we screened a chemical library of clinically approved drugs (1979 compounds) by high-throughput screening and showed that the Ca2+ channel blocker cilnidipine has an inhibitory effect on rodent and human P2X7R. In primary cultured rat microglial cells, cilnidipine inhibited P2X7R-mediated Ca2+ responses and IL-1ß release. Moreover, in a rat model of neuropathic pain, the intrathecal administration of cilnidipine produced a reversal of nerve injury-induced mechanical hypersensitivity, a cardinal symptom of neuropathic pain. These results point to a new inhibitory effect of cilnidipine on microglial P2X7R-mediated inflammatory responses and neuropathic pain, proposing its therapeutic potential.

Synthesis of DFGH-Ring Derivatives of Physalins via One-Pot Construction of GH-Ring and Evaluation of Their NF-κB-Inhibitory Activity.

We designed and synthesized a series of derivatives containing the right-side DFGH-ring structure of physalin-type natural products, decorated with a hydrophobic substituent. The synthetic scheme utilizes a highly efficient, one-pot protocol for simultaneous construction of the GH-ring system, promoted by HF/pyridine. Among the compounds synthesized, 5d inhibited TNF-α-stimulated NF-κB activation with similar potency to physalin B.

MBNL1-Associated Mitochondrial Dysfunction and Apoptosis in C2C12 Myotubes and Mouse Skeletal Muscle.

We explored the interrelationship between a tissue-specific alternative splicing factor muscleblind-like 1 (MBNL1) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), B-cell lymphoma 2 (Bcl-2) or Bcl-2-associated X protein (Bax) in C2C12 myotubes and mouse skeletal muscle to investigate a possible physiological role of MBNL1 in mitochondrial-associated apoptosis of skeletal muscle. Expression level of PGC-1α and mitochondrial membrane potential evaluated by the fluorescence ratio of JC-1 aggregate to monomer in C2C12 myotubes were suppressed by knockdown of MBNL1. Conversely, the ratio of Bax to Bcl-2 as well as the apoptotic index in C2C12 myotubes was increased by MBNL1 knockdown. In plantaris muscle, on the other hand, not only the minimum muscle fiber diameter but also the expression level of MBNL1 and PGC-1α in of 100-week-old mice were significantly lower than that of 10-week-old mice. Furthermore, the ratio of Bax to Bcl-2 in mouse plantaris muscle was increased by aging. These results suggest that MBNL1 may play a key role in aging-associated muscle atrophy accompanied with mitochondrial dysfunction and apoptosis via mediating PGC-1α expression in skeletal muscle.

New algorithm using L1 regularization for measuring electron energy spectra.

Retrieving the spectrum of physical radiation from experimental measurements typically involves using a mathematical algorithm to deconvolve the instrument response function from the measured signal. However, in the field of signal processing known as "Source Separation" (SS), which refers to the process of computationally retrieving the separate source components that generate an overlapping signal on the detector, the deconvolution process can become an ill-posed problem and crosstalk complicates the separation of the individual sources. To overcome this problem, we have designed a magnetic spectrometer for inline electron energy spectrum diagnosis and developed an analysis algorithm using techniques applicable to the problem of SS. An unknown polychromatic electron spectrum is calculated by sparse coding using a Gaussian basis function and an L1 regularization algorithm with a sparsity constraint. This technique is verified by using a specially designed magnetic field electron spectrometer. We use Monte Carlo simulations of the detector response to Maxwellian input energy distributions with electron temperatures of 5.0 MeV, 10.0 MeV, and 15.0 MeV to show that the calculated sparse spectrum can reproduce the input spectrum with an optimum energy bin width automatically selected by the L1 regularization. The spectra are reproduced with a high accuracy of less than 4.0% error, without an initial value. The technique is then applied to experimental measurements of intense laser accelerated electron beams from solid targets. Our analysis concept of spectral retrieval and automatic optimization of energy bin width by sparse coding could form the basis of a novel diagnostic method for spectroscopy.

Involvement of exchange protein directly activated by cAMP and tumor progression locus 2 in IL-1ß production in microglial cells following activation of ß-adrenergic receptors.

Endogenous noradrenaline (NA) has multiple bioactive functions and, in the central nervous system (CNS), has been implicated in modulating neuroinflammation via ß-adrenergic receptors (ß-ARs). Microglia, resident macrophages in the CNS, have a central role in the brain immune system and have been reported to be activated by NA. However, intracellular signaling mechanisms of the AR-mediated proinflammatory responses of microglia are not fully understood. Using a rapid and stable in vitro reporter assay system to evaluate IL-1ß production in microglial BV2 cells, we found that NA and the ß-AR agonist isoproterenol upregulated the IL-1ß reporter activity. This effect was suppressed by ß-AR antagonists. We further examined the involvement of EPAC (exchange protein directly activated by cAMP) and TPL2 (tumor progression locus 2, MAP3K8) and found that inhibitors for EPAC and TPL2 reduced AR agonist-induced IL-1ß reporter activity. These inhibitors also suppressed NA-induced endogenous Il1b mRNA expression and IL-1ß protein production. Our results suggest that EPAC and TPL2 are involved in ß-AR-mediated IL-1ß production in microglial cells, and extend our understanding of its intracellular signaling mechanism.

Gemcitabine induces Parkin-independent mitophagy through mitochondrial-resident E3 ligase MUL1-mediated stabilization of PINK1.

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis. PTEN-induced kinase (PINK1), a key regulator of mitophagy, is degraded constitutively under steady-state conditions. During mitophagy, it becomes stabilized in the outer mitochondrial membrane, particularly under mitochondrial stress conditions, such as in treatment with uncouplers, generation of excessive mitochondrial reactive oxygen species, and formation of protein aggregates in mitochondria. Stabilized PINK1 recruits and activates E3 ligases, such as Parkin and mitochondrial ubiquitin ligase (MUL1), to ubiquitinate mitochondrial proteins and induce ubiquitin-mediated mitophagy. Here, we found that the anticancer drug gemcitabine induces the stabilization of PINK1 and subsequent mitophagy, even in the absence of Parkin. We also found that gemcitabine-induced stabilization of PINK1 was not accompanied by mitochondrial depolarization. Interestingly, the stabilization of PINK1 was mediated by MUL1. These results suggest that gemcitabine induces mitophagy through MUL1-mediated stabilization of PINK1 on the mitochondrial membrane independently of mitochondrial depolarization.