ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes.

This study aimed to investigate the potential role of pyruvate kinase M2 (PKM2) and extracellular regulated protein kinase (ERK) in arsenic-induced cell proliferation. L-02 cells were treated with 0.2 and 0.4 µmol/L As3+, glycolysis inhibitor (2-deoxy-D-glucose,2-DG), ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene, U0126] or transfected with PKM2 plasmid. Cell viability, proliferation, lactate acid production, and glucose intake capacity were determined by CCK-8 assay, EdU assay, lactic acid kit and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake kit, respectively. Also, levels of PKM2, phospho-PKM2S37, glucose transporter protein 1 (GLUT1), lactate dehydrogenase A (LDHA), ERK, and phospho-ERK were detected using Western blot and the subcellular localization of PKM2 in L-02 cells was detected by immunocytochemistry (ICC). Treatment with 0.2 and 0.4 µmol/L As3+ for 48 h increased the viability and proliferation of L-02 cells, the proportion of 2-NBDG+ cell and lactic acid in the culture medium, and GLUT1, LDHA, PKM2, phospho-PKM2S37, and phospho-ERK levels and PKM2 in nucleus. Compared with the 0.2 µmol/L As3+ treatment group, the lactic acid in the culture medium, cell proliferation and cell viability, and the expression of GLUT1 and LDHA were reduced in the group co-treated with siRNA-PKM2 and arsenic or in the group co-treated with U0126. Moreover, the arsenic-increased phospho-PKM2S37/PKM2 was decreased by U0126. Therefore, ERK/PKM2 plays a key role in the Warburg effect and proliferation of L-02 cells induced by arsenic, and also might be involved in arsenic-induced upregulation of GLUT1 and LDHA. This study provides a theoretical basis for further elucidating the carcinogenic mechanism of arsenic.

Association between arsenic (+3 oxidation state) methyltransferase gene polymorphisms and arsenic methylation capacity in rural residents of northern China: a cross-sectional study.

Arsenic is a toxic metal-like element. The toxic reaction of the body to arsenic is related to the ability of arsenic methylation metabolism. As the rate-limiting enzyme of arsenic methylation metabolism, the genetic single nucleotide polymorphisms (SNPs) of arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene are related to capacity of arsenic methylation. In this paper, we investigated the association of five SNPs (rs7085104, rs3740390, 3740393, rs10748835, and rs1046778) in AS3MT with arsenic methylation metabolizing using the data and samples from a cross-sectional case-control study of arsenic and Type 2 diabetes mellitus conducted in Shanxi, China. A total of 340 individuals were included in the study. Urinary total arsenic (tAs, µg/L) was detected by liquid chromatography-atomic fluorescence spectrometry (LC-AFS). According to "safety guidance value of urinary arsenic for population" as specified in WS/T665-2019 (China), participants were divided into the control group (tAs ≤ 32 µg/L, n = 172) and arsenic-exposed group (tAs > 32 µg/L, n = 168). iAs%, MMA%, and DMA% are as the indicator of arsenic methylation capacity. The genotypes of AS3MT SNPs were examined by Multiple PCR combined sequencing. Linear regression analysis showed that AG + GG genotype in rs7085104 was associated with decreased iAs% and increased DMA%. Moreover, AG + AA genotype in rs10748835 and TC + CC genotype in rs1046778 were associated with decreased iAs% and MMA% and increased DMA%. The interaction between rs7085104 and arsenic is associated with iAs% and DMA%. The interaction of rs3740390 and rs10748835 with arsenic is associated with iAs%. Haplotype CTAC (rs3740393-rs3740390-rs10748835-rs1046778) was associated with lower iAs% and higher DMA%, but this association disappeared after adjusting for age, gender, drink, smoking, BMI and tAs. Haplotype GCAC was associated with decreased MMA%. Our study provides additional support for revealing the factors influencing the metabolic capacity of arsenic methylation and might be helpful to identify the population susceptible to arsenic exposure through individualized screening in the future.

SIRT1/P53 pathway is involved in the Arsenic induced aerobic glycolysis in hepatocytes L-02 cells.

Arsenic is a known human carcinogen. Low doses of arsenic can induce cell proliferation, but the mechanism remains elusive. Aerobic glycolysis, also known as the Warburg effect, is one of the characteristics of tumour cells and rapidly proliferating cells. P53 is a tumour suppressor gene that has been shown to be a negative regulator of aerobic glycolysis. SIRT1 is a deacetylase that inhibits the function of P53. In this study, we found that P53 was involved in low dose of arsenic-induced aerobic glycolysis through regulating HK2 expression in L-02 cells. Moreover, SIRT1 not only inhibited P53 expression but also decreased the acetylation level of P53-K382 in arsenic-treated L-02 cells. Meanwhile, SIRT1 influenced the expression of HK2 and LDHA, which then promoted arsenic-induced glycolysis in L-02 cells. Therefore, our study demonstrated that the SIRT1/P53 pathway is involved in arsenic-induced glycolysis, thereby promoting cell proliferation, which provides theoretical basis for enriching the mechanism of arsenic carcinogenesis.

Peptide-assembled nanoparticles targeting tumor cells and tumor microenvironment for cancer therapy.

Tumor cells and corrupt stromal cells in the tumor microenvironment usually overexpress cancer-specific markers that are absent or barely detectable in normal cells, providing available targets for inhibiting the occurrence and development of cancers. It is noticeable that therapeutic peptides are emerging in cancer therapies and playing more and more important roles. Moreover, the peptides can be self-assembled and/or incorporated with polymeric molecules to form nanoparticles via non-covalent bond, which have presented appealing as well as enhanced capacities of recognizing targeted cells, responding to microenvironments, mediating internalization, and achieving therapeutic effects. In this review, we will introduce the peptide-based nanoparticles and their application advances in targeting tumor cells and stromal cells, including suppressive immune cells, fibrosis-related cells, and angiogenic vascular cells, for cancer therapy.

Malignant growth of arsenic-transformed cells depends on activated Akt induced by reactive oxygen species.

Arsenic is an identified carcinogen for humans.In this study, chronic exposure of human hepatocyte L-02 to low-doses of inorganic arsenic caused cell malignant proliferation. Meanwhile, compared with normal L-02 cells, arsenic-transformed malignant cells, L-02-As displayed more ROS and significantly higher Cyclin D1 expression as well as aerobic glycolysis. Moreover, Akt activation is followed by the upregulation of Cyclin D1 and HK2 expression in L-02-As cells, since inhibition of Akt activity by Ly294002 attenuated the colony formation in soft agar and decreased the levels of Cyclin D1 and HK2. In addition, scavenging of ROS by NAC resulted in a decreased expression of phospho-Akt, HK2 and Cyclin D1, and attenuates the ability of anchorage-independent growth ofL-02-As cells, suggested that ROS mediated the Akt activation in L-02-As cells. In summary, our results demonstrated that ROS contributes to the malignant phenotype of arsenic-transformed human hepatocyte L-02-As via the activation of Akt pathway.

Nanomicelles co-loading CXCR4 antagonist and doxorubicin combat the refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is featured with poor prognosis and high mortality, because chemo-resistance, nonspecific distribution and dose-limiting toxicity lead to a high rate of relapse and a very low 5-year survival percentage of less than 25%. CXCR4 is a highly expressed chemokine receptor in multiple types of AML cells and closely associated with the drug resistance and relapse. In this work, we integrate a chemically synthesized CXCR4 antagonistic peptide and doxorubicin using DSPE-mPEG2000 micelles (referred to as M-E5-Dox) that is applied to a very challenging refractory AML mouse model as well as human AML cell lines. Results showed that M-E5-Dox can effectively bind to the CXCR4-expressing AML cells, downregulating the signaling proteins mediated by CXCR4/CXCL12 axis and increasing the cellular uptake of Dox. Importantly, M-E5-Dox remarkably decreases the leukemic cells in the peripheral blood and bone marrow, as well as their infiltration in the spleen and liver of the AML mice, which in turn prolongs the survival significantly. Meanwhile, M-E5-Dox did not increase the cardiotoxicity of Dox. In conclusion, M-E5-Dox harnesses the functions of CXCR4 specific binding and CXCR4 antagonism of the peptide and the tumor cell killing capacity of Dox, which displays significant therapeutic effects and promising translational potentials for the treatment of refractory AML.

The ROS/NF-κB/HK2 axis is involved in the arsenic-induced Warburg effect in human L-02 hepatocytes.

Arsenic has been identified as a carcinogen, although the molecular mechanism underlying itscarcinogenesis has not been fully elucidated. To date, only a few studies have attempted to confirm a direct link between oxidative stress and the Warburg effect . This study demonstrated that 0.2 µmol/L As3+ induced the Warburg effect to contribute to abnormal proliferation of L-02 cells, that was mediated by upregulation of hexokinase 2 (HK2), a key enzyme in glycolysis. Further study indicated that arsenic-induced accumulation of reactive oxygen species (ROS) activated the nuclear factor kappa B (NF-κB) signaling pathway by phosphorylation of p65 at the Ser536 and Ser276 sites, leading to upregulated expression of HK2. We therefore concluded that the ROS/NF-κB/HK2 axis contributes to the Warburg effect and cell proliferation induced by low doses of arsenic.AbbreviationsROS, Reactive oxygen species; NAC, N-acetyl-L-cysteine; 2-DG, 2-deoxy-D-glucose; 2-NBDG, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose.

CD123 Antagonistic Peptides Assembled with Nanomicelles Act as Monotherapeutics to Combat Refractory Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Due to the development of drug resistance to traditional chemotherapies and high relapse rate, AML still has a low survival rate and there is in an urgent need for better treatment strategies. CD123 is widely expressed by AML cells, also associated with the poor prognosis of AML. In this study, we fabricated nanomicelles loaded with a lab-designed CD123 antagonistic peptide, which were referred to as mPO-6. The antagonistic and therapeutic effects were investigated with CD123+ AML cell lines and a refractory AML mouse (AE and CKITD816V) model. Results show that mPO-6 can specifically bind to the CD123+ AML cells and inhibit the cell viability effectively. Intravenous administration of mPO-6 significantly reduces the percentage of AML cells' infiltration and prolongs the median survival of AML mice. Further, the efficiency of mPO-6 is demonstrated to interfere with the axis of CD123/IL-3 via regulating the activation of STAT5, PI3K/AKT, and NF-κB signaling pathways related to cell proliferation or apoptosis at the level of mRNA and protein in vivo and in vitro. In conclusion, the novel CD123 antagonistic peptide micelle formulation mPO-6 can significantly enhance apoptosis and prolong the survival of AML mice by effectively interfering with the axis of CD123/IL-3 and therefore is a promising therapeutic candidate for the treatment of refractory AML.

Low-dose arsenic trioxide enhances membrane-GLUT1 expression and glucose uptake via AKT activation to support L-02 cell aberrant proliferation.

Long term low dose exposure of arsenic has been reported to lead various cells proliferation and malignant transformation. GLUT1, as the key transporter of glucose, has been reported to have association with rapid proliferation of various cells or tumor cells. In our study, we found that low dose exposure to arsenic trioxide (0.1µmol/L As2O3) could induce an increase in glucose uptake and promote cell viability and DNA synthesis. And, 2-DG, a non-metabolized glucose analog, significantly decreased the glucose uptake and cell proliferation of 0.1µmol/L As2O3 treated L-02 cells. However, 4 mmol/L 2-DG was co-utilized with equal dose glucose had no significant effect on the cell proliferation of 0.1µmol/L As2O3 treated L-02 cells. Further studies showed that exposure to 0.1µmol/L As2O3 could promote the expression of GLUT1 on plasma membrane. Inhibition of GLUT1 expression by 5µmol/L BAY-876 significantly decreased the abilities of glucose uptake and cell proliferation in As2O3-treated L-02 cells. Moreover, 0.1µmol/L As2O3 induced the AKT activation indicated by increased the phospho-AKT (Ser473 and Thr308). Knockdown AKT by shRNA or inhibited AKT activation by LY294002 was followed by significantly decreased glucose uptake, GLUT1 plasma membrane expression and cell proliferation in As2O3-treated L-02 cells. All in all, these results demonstrated that arsenic trioxide-induced AKT activation contributed to the cells proliferation through upregulating expression of GLUT1 on plasma membrane that enhanced glucose uptake.

Arsenic exposure elevated ROS promotes energy metabolic reprogramming with enhanced AKT-dependent HK2 expression.

Exposure to inorganic or organic arsenic compounds continues to pose substantial public health concerns for hundreds of millions of people around the globe. Highly exposed individuals are susceptible to various illnesses, including impairments and cancers of the lung, liver, skin and bladder. Long-term exposure to low-dose arsenic has been identified to induce aerobic glycolysis, which contributes to cells aberrant proliferation. However, the mechanism underlying arsenic-induced aerobic glycolysis is still unclear. Here, mtDNA copy number is enhanced in arsenic-exposed populations and a positive correlation between serum HK2 and urinary total arsenic was observed in the individuals with high urine arsenic (≥ 0.032 mg/L). In a rat model of trivalent arsenic (iAs3+) exposure, the levels of HK2, NDUFA9 and NDUFB8 were increased in the rats treated with iAs3+ daily by gavage for 12 weeks than those in the control rats. Subsequently, in a low-dose arsenic exposure cell model we found that 0.2 µmol/L iAs3+ induced aerobic glycolysis to promote L-02 cells proliferation and inhibit apoptosis, in which HK2 played an important role. Further studies showed accumulated ROS determined the metabolic reprogramming via activating AKT and then increasing HK2 expression. On the one hand, activated AKT induced aerobic glycolysis by increasing HK2 to promote L-02 cells viability and DNA synthesis; on the other hand, phosphorylated AKT induced HK2 mitochondrial outer-membrane location with VDAC1 to inhibit apoptosis. Taken together, our results indicated that ROS induced by low-dose arsenic exposure determined energy metabolic reprogramming and acted a critical regulator for AKT-dependent HK2 expression and aerobic glycolysis.

miR-21-5p and canonical Wnt signaling pathway promote osteoblast function through a feed-forward loop induced by fluoride.

Long-term excessive exposure to fluoride from environmental sources can cause serious public health problems such as dental fluorosis and skeletal fluorosis. The aberrant activation of osteoblasts in the early stage is one of the critical steps during the pathogenesis of skeletal fluorosis and canonical Wnt signaling pathway participate in the progress. However, the specific mechanism that how canonical Wnt signaling pathway was mediated is not yet clear. In this study, we found that miR-21-5p induced the activation of canonical Wnt signaling pathway via targeting PTEN and DKK2 during fluoride induced osteoblasts activation and firstly demonstrated the forward loop between canonical Wnt signaling and miR-21-5p in the process. These findings suggested an important regulatory role of miR-21-5p on canonical Wnt signaling pathway during skeletal fluorosis and miR-21-5p might be a potential therapeutic target for skeletal fluorosis.

Iron oxide nanoparticles cause surface coating- and core chemistry-dependent endothelial cell ferroptosis.

Iron oxide nanoparticles (IONPs) are mostly intended to be administrated intravenously, understanding the interaction of IONPs with vascular endothelial cells is extremely crucial for developing safe application regimes of IONPs. In this work, interactions of three kinds of IONPs to endothelial cells were investigated both in human umbilical vein endothelial cells (HUVECs) and in healthy mice. Both meso-2,3-dimercaptosuccinic acid (DMSA) coated Fe3O4 NPs (DMSA-Fe3O4 NPs) and DMSA-Fe2O3 NPs induced cell growth inhibition, while polyglucose sorbitol carboxymethyether coated Fe2O3 NPs(PSC-Fe2O3 NPs) did not. The PSC coating inhibited the cellular uptake of the IONPs. Both DMSA-Fe3O4 and DMSA-Fe2O3 NPs induced ferroptosis of HUVEC through upregulating phospholipid peroxides, which could be inhibited by typical ferroptosis inhibitors ferrostatin-1, Trolox and deferoxamine. Moreover, transforming growth factor beta 1 (TGFß1) was upregulated by DMSA-Fe3O4 NPs at protein and gene level. The inhibitor of TGFß1 receptor LY210 could reduce the effect. When being intravenously injected in mice, DMSA-Fe3O4 NPs were observed locating in the liver, increased the levels of lipid peroxidation (4-hydroxynonenal), acyl-CoA synthetase long-chain family member 4(ACSL4) and TGFß1, indicating ferroptosis occurrence in vivo. The ferroptosis of vascular endothelial cells in exposure with IONPs depended on the surface coating and core chemistry of the NPs. Both DMSA-Fe3O4 NPs and DMSA-Fe2O3 NPs could induce the ferroptosis of endothelial cells, while PSC-Fe2O3 NPs did not induce ferroptosis and apoptosis possibly due to the very low cellular uptake. DMSA-Fe3O4 NPs and TGFß1 formed feedforward loop to induce ferroptosis.

A novel CD123-targeted therapeutic peptide loaded by micellar delivery system combats refractory acute myeloid leukemia.

Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival rate due to the refractory feature and high rate of relapse. CD123 is highly expressed on multiple types of AML cells, especially leukemia stem cells, and closely associated with the poor prognosis of AML. Aiming to meet the urgent demand to targeted therapeutics for the refractory AML patients, herein we synthesize a CD123 antagonistic peptide (PO-6) loaded in nanomicelles (mPO-6), and investigated its therapeutic effect and pharmacokinetics on a lab-established refractory AML mice model (AE & CKITD816V). It is shown that the PO-6 can effectively bind to the CD123+ AML cells and the micellar formulation mPO-6 increases the dissolution stability and the specific binding capacity. When injected intravenously, mPO-6 significantly prolongs the survival of the refractory AML mice by interfering CD123/IL-3 axis, evidenced by the down regulation of phosphorylation of STAT5 and PI3K/AKT and the inhibition of activated NF-κB in the nucleus, as well as by the analysis results of next generation RNA-sequencing (RNA-seq) with the bone marrow of the AML mice. The antagonistic effect leads to the significantly reduction of AML cells infiltration in the bone marrow of the AML mice. In conclusion, mPO-6 could provide a potent antagonistic therapeutic approach for targeted treatment of AML.

High Iodine Induces the Proliferation of Papillary and Anaplastic Thyroid Cancer Cells via AKT/Wee1/CDK1 Axis.

High iodine can alter the proliferative activity of thyroid cancer cells, but the underlying mechanism has not been fully elucidated. Here, the role of high iodine in the proliferation of thyroid cancer cells was studied. In this study, we demonstrated that high iodine induced the proliferation of BCPAP and 8305C cells via accelerating cell cycle progression. The transcriptome analysis showed that there were 295 differentially expressed genes (DEGs) in BCPAP and 8305C cells induced by high iodine, among which CDK1 expression associated with the proliferation of thyroid cancer cells induced by high iodine. Moreover, the western blot analysis revealed that cells exposed to high iodine enhanced the phosphorylation activation of AKT and the expression of phospho-Wee1 (Ser642), while decreasing the expression of phospho-CDK1 (Tyr15). Importantly, the inhibition of AKT phosphorylation revered the expression of CDK1 induced by high iodine and arrested the cell cycle in the G1 phase, decreasing the proliferation of thyroid cancer cells induced by high iodine. Taken together, these findings suggested that high iodine induced the proliferation of thyroid cancer cells through AKT-mediated Wee1/CDK1 axis, which provided new insights into the regulation of proliferation of thyroid cancer cells by iodine.

Prussian blue nanoparticles induce myeloid leukemia cells to differentiate into red blood cells through nanozyme activities.

Numerous types of diseases cause serious anemia, which is characterized by a significantly decreased number of circulating red blood cells. The key reason is retarded terminal erythroid differentiation, which is largely involved in the downregulation of intracellular reactive oxygen species (ROS) and insufficient iron uptake. Prussian blue nanoparticles (PBNPs) have been demonstrated to be capable of scavenging ROS via multienzyme-like activity and contain the iron element. The aim of this study was to figure out whether PBNPs can induce terminal erythroid differentiation in myeloid leukemia cells K562 and to investigate the underlying mechanisms. Our results showed that PBNPs were taken up by K562 cells, which reduced the intracellular ROS level in the cells, upregulated the late erythroid surface marker GYPA (CD235a) and downregulated the early erythroid surface marker TFRC (CD71), clearly indicating the occurrence of terminal erythroid differentiation. In addition, the cells became smaller in size after incubation with PBNPs, providing strong side evidence that the cells had undergone terminal differentiation. Mechanistic studies indicated that PBNP-induced terminal differentiation was associated with the upregulation of the nuclear transcriptional factor NFE2 and downregulation of GATA1, both of which are closely related to the variation of intracellular ROS levels. In conclusion, PBNPs demonstrated a novel function by effectively inducing terminal erythroid differentiation in myeloid leukemia cells, which is of great significance in improving the blood profiles of anemia patients.

A Chinese family with familial hemiplegic migraine type 2 due to a novel missense mutation in ATP1A2.

BACKGROUND: ATP1A2 has been identified as the genetic cause of familial hemiplegic migraine type 2. Over 80 ATP1A2 mutations have been reported, but no data from Chinese family studies has been included. Here, we report the first familial hemiplegic migraine type 2 Chinese family with a novel missense mutation. METHODS: Clinical manifestations in the family were recorded. Blood samples from patients and the unaffected members were collected for whole-exome sequencing to identify the pathogenic mutation. Seven online softwares (SIFT, PolyPhen-2, PROVEAN, PANTHER, MutationTaster2, MutationAssessor and PMut) were used for predicting the pathogenic potential of the mutation. PredictProtein, Jpred 4 and PyMOL were used to analyze structural changes of the protein. The mutation function was further tested by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. RESULTS: All patients in the family had typical hemiplegic migraine attacks. Co-segregation of the mutation with the migraine phenotype in four generations, with 10 patients, was completed. The identified novel mutation, G762S in ATP1A2, exhibited the disease-causing feature by all the predictive softwares. The mutation impaired the local structure of the protein and decreased cell viability. CONCLUSION: G762S in ATP1A2 is a novel pathogenic mutation identified in a Chinese family with familial hemiplegic migraine, which causes loss of function by changing the protein structure of the Na+/K+-ATPase α2 subunit.

Facile Synthesis and Cytotoxicity of Phenazine-Chromene Hybrid Molecules Derived from Phenazine Natural Product.

AIM AND OBJECTIVE: Small molecule targeted drugs can effectively reduce the toxicity and side effects of drugs, and improve the efficacy of drugs by their specific antitumor activity. Hence, the development of small molecular targeted drugs for cancer has important significance. This study was undertaken to design and synthesize novel phenazine-chromene hybrid molecules in order to optimize the structure and improve the efficacy of this kind of hybrids. MATERIALS AND METHODS: O-diaminobenzene was used as starting material to synthesize twentyfour heterocyclic compounds designed as hybrid molecules of phenazine and 4H-chromene pharmacophores by facile methods. The structures of the compound were confirmed by 1H NMR, 13C NMR and HRMS. Furthermore, the synthesized compounds were evaluated for in vitro activity against four human cancer cell lines and two non-cancer cell lines by MTT test. RESULTS: Some compounds showed strong cytotoxic activities against HepG2 and A549 cancer lines (IC50 = 5-10 µM). Comparing 2i with 2l, the introduction of hydrophilic groups on the phenazine core could not improve the antiproliferative activity significantly. Except 2d and 3c, compounds owning chlorine substituent on the 4H-chromene pharmacophore seemingly contribute to enhance the compounds' antiproliferative activity. Specially, compound 3c showed highest cytotoxicity against A549 cells with IC50 values of 3.3±0.4 µM. Furthermore, all compounds showed low or no cytotoxicity against HUVEC and L02 non-cancer cells in vitro. CONCLUSION: Compound 3c may be used as potential lead molecule against A549 cancer cells.

Leukoencephalopathy with cerebral calcification and cysts: Cases report and literature review.

BACKGROUND: Leukoencephalopathy with calcifications and cysts (LCC) is a rare disease in which parenchymal cysts and calcifications within a widespread leukoencephalopathy can cause a broad spectrum of neurological symptoms. We present cases with adult LCC and discuss previously described entities in relevant literature. CASE PRESENTATION: Two cases of adult-onset LCC confirmed by clinical presentations, typical neuroimaging and neuropathological findings are reported. LITERATURE REVIEW: A detailed search of all relevant reports published in the English language between 1996 and 2015 via PubMed (http://www.ncbi.nlm.nih.gov/pubmed) was performed, with "Leukoencephalopathy", "cerebral calcifications" and "cysts" as keywords. Including the current cases, we summarized the clinical presentations, neuroimaging features, biopsy features, and genetic features of 38 LCC patients. CONCLUSION: Our findings suggested that LCC could be diagnosed by clinical presentations, neuroimaging and gene detection, and biopsy might not be necessary. Therefore, we propose a diagnostic flow chart for neuroimaging in leukoencephalopathy, cerebral calcifications and cysts.