OBJECTIVE: Aloin, the main active component in Aloe vera (L.) Burm. f., has shown promising anti-tumor effects. This study investigated the impact of aloin in lung squamous cell carcinoma (LUSC) and explored its functional mechanism. METHODS: We analyzed the viability, migration, invasion, proliferation, and apoptosis of two LUSC cell lines after treatment with aloin. Target molecules of aloin and downstream target transcripts of nuclear receptor subfamily 3 group C member 2 (NR3C2) were predicted by bioinformatics. The biological functions of NR3C2 and metallothionein 1 M (MT1M) in the malignant properties of LUSC cells were determined. A co-culture system of LUSC cells with monocyte-derived macrophages was constructed. Mouse xenograft tumor models were generated to analyze the functions of aloin and NR3C2 in the tumorigenic activity of LUSC cells and macrophage polarization in vivo. RESULTS: Aloin suppressed malignant properties of LUSC cells in vitro. However, these effects were negated by the silencing of NR3C2. NR3C2 was found to activate MT1M transcription by binding to its promoter. Additional upregulation of MT1M suppressed the malignant behavior of LUSC cells augmented by NR3C2 silencing. Analysis of the M1 and M2 markers/cytokines in the macrophages or the culture supernatant revealed that aloin treatment or MT1M overexpression in LUSC cells enhanced M1 polarization while suppressing M2 polarization of macrophages, whereas NR3C2 silencing led to reverse trends. Consistent findings were reproduced in vivo. CONCLUSION This study demonstrated that aloin activates the NR3C2/MT1M axis to suppress the malignant behavior of LUSC cells and M2 macrophage polarization. Please cite this article as: Chen YN, Lu JY, Gao CF, Fang ZR, Zhou Y. Aloin blocks the malignant behavior of lung squamous cell carcinoma cells and M2 macrophage polarization by modulating the NR3C2/MT1M axis. J Integr Med. 2025; 23(2): 195-208.
Lung Neoplasms/metabolism* ; Humans ; Animals ; Cell Line, Tumor ; Carcinoma, Squamous Cell/metabolism* ; Mice ; Macrophages/drug effects* ; Emodin/analogs & derivatives* ; Metallothionein/genetics* ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; Apoptosis/drug effects* ; Receptors, Glucocorticoid/genetics*

BACKGROUNDDysregulated metallothionein 2A (MT2A) has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer (GC) and its correlation with prognosis.

METHODSReverse transcription-polymerase chain reaction and real-time polymerase chain reaction were used to detect the mRNA expression of MT2A in 12 GC cell lines, normal gastric epithelial GES-1 cells, and 36 GC and adjacent normal tissues. MT2A protein expression was determined in 258 GC tissues and 171 adjacent normal tissues by immunohistochemistry.

RESULTSMT2A mRNA expression was lower in GC cells and primary tumors than in GES-1 cells and adjacent normal tissues, respectively. High protein expression of MT2A was present in 130 of 171 normal tissues (76.0%) and in 56 of 258 GC tissues (21.7%; P < 0.001). MT2A protein expression was higher in well/moderately differentiated GC (22/54; 40.7%) than in poorly differentiated GC (34/204; 16.7%; P < 0.001). Moreover, the protein expression of MT2A was lower in diffuse-type GC (6/82; 7.3%) than in intestinal-type GC (50/176; 28.4%; P = 0.0001). Importantly, MT2A expression was an independent prognostic factor for GC, and decreased MT2A expression was associated with poor clinical outcome (P < 0.001). The expression status of MT2A could predict prognosis in intestinal and diffuse-type GCs.

CONCLUSIONExpression status of MT2A might be a useful prognostic biomarker for GC, especially when used in combination with Lauren's classification.

Adult ; Aged ; Cell Line, Tumor ; Female ; Humans ; Logistic Models ; Male ; Metallothionein ; analysis ; genetics ; MicroRNAs ; analysis ; Middle Aged ; Prognosis ; Proportional Hazards Models ; Stomach Neoplasms ; chemistry ; classification ; pathology

OBJECTIVETo investigate anticancer effect and potential mechanism of tanshinone II(A) (Tan II(A)) on human nasopharyngeal carcinoma cell line CNE cells.

METHODAntiproliferative effect of Tan II(A) on CNE cells was evaluated by morphological examination, cell growth curves, colonial assay and MTT assay. Apoptosis detection was carried out using Hoechest 33258 and PI double-dyeing method. Intracellular Ca2+ concentration and mitochondria membrane potential were detected by fluorospectrophotometer. Bad and MT-1A transcript analysis in CNE cells was analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTTan II(A) could inhibit CNE cells proliferation in dose- and time-dependent manner. 50% inhibiting concentration of Tan II(A) on CNE cells in 24, 48, 72 h was 45.7, 24.8, 3.3 mg x L(-2), respectively. Typical apoptotic morphology such as chromatin aggregation was observed in CNE cells with Tan II(A) treated for 24 h, and the apoptotic inducing effect was in a dose-dependent manner. After treated with Tan II(A), intracellular Ca2+ concentration of CNE cells was increased, mitochondria membrane potential of the cells was decreased, relative mRNA level of Bad and MT-1A was up-regulated.

CONCLUSIONTan II(A) had anticancer effect on CNE cells through apoptosis via calcineurin-dependent pathway and MT-1A downregulation.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Calcium ; metabolism ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Diterpenes, Abietane ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Metallothionein ; genetics ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Signal Transduction ; drug effects ; bcl-Associated Death Protein ; genetics

Metallothioneins (MTs) are a group of intracellular proteins of low molecular weight and rich cysteine, encoded by a family of genes containing at least 10 functional isoforms in human. MTs widely exist in the male reproductive system and are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. This review describes the location and effects of MTs in the male reproductive system.
Gene Expression Profiling ; Genitalia, Male ; metabolism ; physiopathology ; Humans ; Male ; Metallothionein ; genetics ; metabolism ; physiology ; Protein Isoforms ; genetics ; metabolism ; physiology

OBJECTIVETo study the metallothionein genes of Salvia miltiorrhiza through bioinformatics and characterization of tissue expression in regenerated shoots.

METHODMetallothionein genes were obtained by cDNA microarray analyze. BLAST was used for align, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT-PCR method was used to detect the gene expression level.

RESULTTwo metallothionein genes were obtained which were contained a deduced amino acid sequence of 80 and 79 residues, named as SmMT-2a and SmMT-2b, they had a homology of 71.25%. Semiquantitative RT-PCR indicated that metallothionein genes were expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in leaf than in root and stem.

CONCLUSIONIt was the first time that metallothionein genes were obtained from S. miltiorrhiza. It provides a good basis for further functional study of S. miltiorrhiza.

Amino Acid Sequence ; Base Sequence ; DNA, Complementary ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; Genomics ; Metallothionein ; genetics ; metabolism ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Open Reading Frames ; Plants, Medicinal ; genetics ; metabolism ; Salvia miltiorrhiza ; genetics ; metabolism ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid

Metallothioneins (MT) are potential candidates for medicine development and application. For the purpose of expressing recombinant MT in E. coli, a crab MT cDNA cloned into pGEM-T was subcloned into pET-GST and then transformed into Escherichia Coli BL21. The fusion protein was proved to be expressed in both soluble and insoluble form by SDS-PAGE and western blot. Since metallothionein chelate metal ions, which may effects the physiological process of E. coli, caused the production of recombinant protein was lower than expected. Optimization of the ions content in the culture medium improved expression. The protein was purified by Zn2+ affinity chromatography, and rinsed off with high imidazole (1.5 M) which was the result of MT chelating instead of His-tag. This fusion protein laid a foundation of further study on the structural and functional biology of metallothionein.
Animals ; Brachyura ; genetics ; Escherichia coli ; genetics ; metabolism ; Metallothionein ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

People with upper body or visceral obesity have a much higher risk of morbidity and mortality from obesity-related metabolic disorders than those with lower body obesity. In an attempt to develop therapeutic strategies targeting visceral obesity, depot- specific differences in the expression of genes in omental and subcutaneous adipose tissues were investigated by DNA array technology, and their roles in adipocyte differentiation were further examined. We found that levels of metallothionein-II (MT-II) mRNA and protein expression were higher in omental than in subcutaneous adipose tissues. The study demonstrates that MT-II may play an important role in adipocyte differentiation of 3T3L1 preadipocytes, and that N-acetylcysteine (NAC) inhibits the adipocyte differentiation of 3T3L1 cells by repressing MT-II in a time- and dose-dependent manner. Furthermore, the intraperitoneal administration of NAC to rats and mice resulted in a reduction of body weights, and a marked reduction in visceral fat tissues. These results suggest that MT-II plays important roles in adipogenesis, and that NAC may be useful as an anti-obesity drug or supplement.
Viscera/drug effects/metabolism ; Time Factors ; Subcutaneous Fat/drug effects ; Rats, Sprague-Dawley ; Rats ; Middle Aged ; Mice, Inbred C57BL ; Mice ; Metallothionein/*genetics/metabolism/physiology ; Male ; Humans ; Female ; Down-Regulation/drug effects/genetics ; Dose-Response Relationship, Drug ; Cell Differentiation/drug effects ; Body Weight/drug effects ; Anti-Obesity Agents/*pharmacology ; Animals ; Aged ; Adipose Tissue/cytology/drug effects/metabolism ; Adipocytes/cytology/drug effects/metabolism ; Acetylcysteine/*pharmacology ; 3T3-L1 Cells

OBJECTIVETo investigate the feasibility of metallothionein (MT) gene expression level in human peripheral blood lymphocytes (HPBLs) as a biomarker in cadmium exposure.

METHODSThe MT gene expression level in HPBLs of workers exposed to cadmium was examined using RT-PCR technique, and the exposure assessment and effect assessment were conducted in exposed workers.

RESULTSThe basal MT-1A, IE, IF, IX and MT-2A expression level in workers exposed to cadmium were significantly higher than those in the control group (P < 0.05). The basal MT-1A, IE, IF, IX and MT-2A expression level would be significantly increased with the increase of the blood cadmium (BCd) level (P < 0.05). There was a trend of increase for the mRNA expression of the basal MT-1A, 1E, IF, IX, MT-2A, especially for the mRNA expression of MT-1A and MT-2A (P < 0.05) with the increase of the level of the urine cadmium (UCd). There was a good dose-response relationship between basal MT-1A expression and UCd. The basal MT-1A, IE, IF, IX and MT-2A expression level were significantly correlated with BCd (P < 0.05) while the basal MT-1A, IF and MT-2A expression level were significantly correlated with UCd (P < 0.05). There were dose-effect relationships of BCd to the basal MT-1E, MT-1F, MT-1X and MT-2X expression level respectively and there were also dose-effect relationships of UCd, beta(2)-MG and the urine metallothionein to the basal MT-1A expression.

CONCLUSIONThe expression of the MT gene isoforms in HPBLs can serve as the biomarker for the cadmium exposure and MT-1A can also serve as the effective biomarkers for the cadmium-induced renal toxicity.

Adult ; Biomarkers ; metabolism ; Cadmium ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Gene Expression ; Humans ; Lymphocytes ; metabolism ; Male ; Metallothionein ; biosynthesis ; genetics ; Occupational Exposure ; Protein Isoforms ; biosynthesis ; RNA, Messenger ; genetics

OBJECTIVETo study the gene expression of metallothionein 1 (MT-1) isoforms in human peripheral blood lymphocytes (HPBLs).

METHODSThe expression of mRNA representing the seven active MT-1 genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium.

RESULTSBasal expressions of MT-1X, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT-1H, 1F, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference (P < 0.05). in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, 1F, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium.

CONCLUSIONSGene expressions of MT-1G, MT-1H, MT-1F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.

Adult ; Biomarkers ; metabolism ; Cadmium ; pharmacology ; Cells, Cultured ; DNA Primers ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Lymphocytes ; metabolism ; Male ; Metallothionein ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo compare the expression of metallothionein (MT) genes and proteins in six human pancreatic cancer cell strains and two human pancreatic cancer drug-resistant cell strains and to explore the relationship between the expression of the MT and pancreatic cancer cell chemo-resistance.

METHODSReverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the MT isoform-specific mRNA, and cadmium/hemoglobin saturation-electrochemistry to determine MT protein levels.

RESULTSMT protein expression in the pancreatic cancer cell strains was encoded by MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1X, and MT-2A genes. The expression of MT proteins was upregulated and MT-1B, MT-1E, MT-1X, MT-2A genes overexpressed in human pancreatic cancer drug-resistant cell lines (P < 0.05).

CONCLUSIONExpressions of MT proteins and genes correlate with the proliferation and chemoresistance of human pancreatic cancer cell strains.

Cell Division ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; Metallothionein ; biosynthesis ; genetics ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured