OBJECTIVE: To investigate the effect of sulforaphane (SFN) on G METHODS: KG1a and KG1cells were treated by different concentrations of SFN for 48 h. Flow cytometry (FCM) was used to analyze the phase distribution of cell cycle. High-throughput sequencing was used to detect the effect of SFN on the expression of cell cycle related genes in KG1a cells. The mRNA expression of P53, P21, CDC2 and CyclinB1 were detected by qPCR. The protein expression of P53, CDC2, P-CDC2 and CyclinB1 were detected by Western blot. RESULTS: Cells in the G CONCLUSION SFN induces leukemia cells to block in G
Cell Cycle ; Humans ; Isothiocyanates/pharmacology* ; Leukemia, Myeloid, Acute ; Mitosis ; Sulfoxides

This study was to investigate the effect of phenylhexyl isothiocyanate (PHI) on drug resistance and sensitivity on K562/A02 cell line to adriamycin (ADM) and to elucidate the possible mechanisms. The inhibition rates of ADM and PHI + ADM on growth of K562/A02 cell line were measured by MTT assay, and K562/A02 cell resistant multiple was calculated. The apoptosis rate of K562/A02 cell line, the changes of intracellular ADM concentrations and MRP1 protein level were detected by flow cytometry (FCM). Intracellular reoxidized GSH level was determined by spectrometric enzyme assay and MRP1 mRNA was assayed by semiquantitative RT-PCR before and after using PHI. The results indicated that the survival rate of K562/A02 cell line decreased with the increasing concentration of PHI. Apoptosis rate increased after treatment in combination with two above drugs, the changes of drug resistance multiple and intracellular ADM level had statistical significance between K562/A02 and K562 cells (p < 0.05), when the concentration of PHI was more than 20 micromol/L. Intracellular GSH level in K562/A02 cell line reduced 5% when 1 microg/ml ADM was used alone, and it increased slightly at first, then decreased when more than 10 micromol/L PHI was used. When more than 20 micromol/L PHI was used in combination with 1 microg/ml ADM, intracellular GSH level in K562/A02 cell line decreased progressively with increasing the concentration of PHI. The expressions of MRP1 mRNA and protein had no statistical significance between K562/A02 and K562 cells (p > 0.05) after or before PHI was used. It is concluded that the cyto-toxicity of PHI to K562/A02 cell line does not associate with the depletion of the intracellular GSH. PHI not only enhances the sensitivity of K562/A02 cell line to ADM, but also partially reverses effect of K562/A02 cell resistance to ADM. ADM combined with PHI can diminish side effect and dosage of ADM.
Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Isothiocyanates ; pharmacology ; K562 Cells

OBJECTIVETo investigate the effects of PHI on histone acetylation and methylation in hepatocellular carcinoma line SMMC-7721 cells.

METHODSApoptosis was measured by TUNNEL assay. Histone methylation and acetylation were detected by Western blot.

RESULTSPHI inhibited cells growth and induced apoptosis. PHI treatment resulted in increased acetylation of histone H3 and H4 , elevated level of histone H3 lysine 4 methylation, and decreased level of histone H3 lysine 9 methylation.

CONCLUSIONSPHI can modulate both histone acetylation and methylation, which could remodel chromatin structure. PHI may be a novel anticancer drug.

Acetylation ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Histones ; metabolism ; Humans ; Isothiocyanates ; pharmacology ; Methylation

This study was purposed to investigate the effects of phenylhexyl isothiocyanate (PHI) on Burkitt lymphoma Daudi cell line and regulation of histone acetylation and methylation in Daudi cells, and to explore the potential mechanism. The apoptotic rate of Daudi cells treated with PHI was measured by flow cytometry, the changes of histone H3 and H4 acetylation, histone H3K9 and H3K4 methylation in Daudi cells treated with PHI were detected by Western blot. The results showed that PHI could induce apoptosis of Daudi cells, increased the acetylation level of H3 and H4, enhanced the methylation of H3K4, but reduced the methylation of H3K9. It is concluded that the PHI can up-regulate the acetylation level of histone H3 associated with transcription stimulation and the methylation of histone H3K4, down-regulate the methylation on histone H3K9 associated with transcription inhibition, promotes the apoptosis of Daudi cells. PHI may be a potential agent for target therapy of lymphoma.
Acetylation ; Apoptosis ; drug effects ; Burkitt Lymphoma ; genetics ; metabolism ; Cell Line, Tumor ; Histones ; genetics ; metabolism ; Humans ; Isothiocyanates ; pharmacology ; Methylation

This study was purposed to investigate whether phenylhexyl isothiocyanate (PHI) can reduce p16 gene methylation level or not. The myeloma U226 cell line was cultured with PHI of 0, 5, 10 micromol/L for 72 hours, then DNA was extracted. Hydrosulfite was used to treat the genome DNA of healthy adult, PCR amplification was carried out by using this DNA as template. The gene chip detecting methylation changes of 3 CpG in promoter region of p16 gene was constructed by designing a pair of probes which contain one methylated and one unmethylated probes. This pair of probes was used to detect 3 consecutive sites of CpG island in p16 gene. The standard curve was constructed by using gene chip after the methylated and unmethylated DNA were mixed at different ratio. Then treated samples of U266 cells were dotted on gene chip, obtained results were compared with standard curve to get the quantitative results. The results indicated that the probes on chip had excellent reproducible ability and precision, the methylation level of p16 gene in U266 cells treated with 0, 5 and 10 micromol/L of PHI was determined as 78.2%, 61.7% and 54.8%, respectively. It is concluded that the PHI can reduce the methylation level of p16 gene in U266 cells.
Cell Line, Tumor ; CpG Islands ; DNA Methylation ; Down-Regulation ; Genes, p16 ; Humans ; Isothiocyanates ; pharmacology ; Oligonucleotide Array Sequence Analysis

OBJECTIVE: Acute liver failure (ALF) is characterized by severe liver dysfunction, rapid progression and high mortality and is difficult to treat. Studies have found that sulforaphane (SFN), a nuclear factor E2-related factor 2 (NRF2) agonist, has anti-inflammatory, antioxidant and anticancer effects, and has certain protective effects on neurodegenerative diseases, cancer and liver fibrosis. This paper aimed to explore the protective effect of SFN in ALF and it possible mechanisms of action. METHODS: Lipopolysaccharide and D-galactosamine were used to induce liver injury in vitro and in vivo. NRF2 agonist SFN and histone deacetylase 6 (HDAC6) inhibitor ACY1215 were used to observe the protective effect and possible mechanisms of SFN in ALF, respectively. Cell viability, lactate dehydrogenase (LDH), Fe²⁺, glutathione (GSH) and malondialdehyde (MDA) were detected. The expression of HDAC6, NRF2, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) were detected by Western blotting and immunofluorescence. RESULTS: Our results show that NRF2 was activated by SFN. LDH, Fe²⁺, MDA and ACSL4 were downregulated, while GSH, GPX4 and SLC7A11 were upregulated by SFN in vitro and in vivo, indicating the inhibitory effect of SFN on ferroptosis. Additionally, HDAC6 expression was decreased in the SFN group, indicating that SFN could downregulate the expression of HDAC6 in ALF. After using the HDAC6 inhibitor, ACY1215, SFN further reduced HDAC6 expression and inhibited ferroptosis, indicating that SFN may inhibit ferroptosis by regulating HDAC6 activity. CONCLUSION SFN has a protective effect on ALF, and the mechanism may include reduction of ferroptosis through the regulation of HDAC6. Please cite this article as: Zhang YQ, Shi CX, Zhang DM, Zhang LY, Wang LW, Gong ZJ. Sulforaphane, an NRF2 agonist, alleviates ferroptosis in acute liver failure by regulating HDAC6 activity. J Integr Med. 2023; 21(5): 464-473.
Humans ; Ferroptosis ; NF-E2-Related Factor 2/genetics* ; Liver Failure, Acute/drug therapy* ; Isothiocyanates/pharmacology* ; Glutathione ; Histone Deacetylase 6

In order to investigate the effects of phenylhexyl isothiocyanate (PHI) on proliferation and apoptosis of multiple myeloma cells RPMI8226 in vitro, the RMPI8226 cells were co-cultured with PHI at various concentrations; the apoptosis of PHI-treated cells was assayed by TUNEL; the cell cycle changes of PHI-treated cells were analyzed by FCM; the mitochondrial potential changes of PHI-treated cells were detected by using a potential sensitive dye JC-1 as probe; the VEGF levels secreted from PHI-treated cells were measured by quantitative sandwich ELISA. The results showed that PHI significantly inhibited RPMI8226 cell proliferation, induced their apoptosis at low concentration (0.5 micromol/L), the inhibitory effect was related to PHI concentration. PHI-treated cells were arrested in G(0)/G(1) phase. The RPMI8226 cells showed shift from red fluorescence to green fluorescence in some concentration-dependent manner, indicating increase of mitochondrial depolarization and potential loss by 3-4-fold as compared with control, after treated RMPI8226 cells with 10 micromol/L of PHI for 48 hours, the VEGF level secreted from RMPI8226 cells significantly decreased, it was 35% of control. It is concluded that the PHI can inhibit cell proliferation, induce cell apoptosis of RMPI8226, the cell apoptosis is associated with mitochondrial depolarization and potential loss, the inhibiting VEGF secretion from RMPI8226 cells by PHI may be one of the reasons causing apoptosis. PHI may be a potential therapeutic drug for multiple myeloma.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Humans ; Isothiocyanates ; pharmacology ; Multiple Myeloma ; metabolism ; pathology ; Vascular Endothelial Growth Factor A ; secretion

The organosulfur compound sulforaphane (SFN; C₆H₁₁NOS₂) is a potent cytoprotective agent promoting antioxidant, anti-inflammatory, antiglycative, and antimicrobial effects in in vitro and in vivo experimental models. Mitochondria are the major site of adenosine triphosphate (ATP) production due to the work of the oxidative phosphorylation (OXPHOS) system. They are also the main site of reactive oxygen species (ROS) production in nucleated human cells. Mitochondrial impairment is central in several human diseases, including neurodegeneration and metabolic disorders. In this paper, we describe and discuss the effects and mechanisms of action by which SFN modulates mitochondrial function and dynamics in mammalian cells. Mitochondria-related pro-apoptotic effects promoted by SFN in tumor cells are also discussed. SFN may be considered a cytoprotective agent, at least in part, because of the effects this organosulfur agent induces in mitochondria. Nonetheless, there are certain points that should be addressed in further experiments, indicated here as future directions, which may help researchers in this field of research.
Animals ; Antioxidants/pharmacology* ; Apoptosis/drug effects* ; Brain/ultrastructure* ; Carbon Monoxide Poisoning/metabolism* ; Cytoprotection ; Humans ; Isothiocyanates/pharmacology* ; Membrane Potential, Mitochondrial/drug effects* ; Mitochondria/metabolism* ; Sulfoxides

OBJECTIVETo investigate the effect of tBHQ and sulforaphane on the protein expression in Nrf2-ARE signaling pathway of Caco2 cells.

METHODSHuman colorectal carcinoma Caco2 cells were treated with 20 micromol/L tBHQ and 5 micromol/L sulforaphane (SFN) respectively. Real time PCR, Western blotting and immunoflourescence staining (IF) were performed to measure the target gene expression.

RESULTSNrf2, AKR1C1 and NQO1 protein expressions were increased time-dependently in Caco2 cells after treatment with tBHQ and SFN. Time-course experiments showed that tBHQ and SFN increased the accumulation of Nrf2, and concomitantly increased the protein levels of AKR1C1 and NQO1. Real-time PCR and Western blotting showed that tBHQ and SFN significantly increased the expression of Nrf2 at 8h after the treatment, and AKR1C1 and NQO1 at 16 h. Confocal microscopy technique showed that Nrf2 accumulated in the nucleus at 6-8 h after treatment with tBHQ. After 1 h treatment with tBHQ the nuclear Nrf2 maintained at elevated level for at least 4 h with tBHQ withdrawn.

CONCLUSIONtBHQ and SFN induced nuclear accumulation of Nrf2 and activated Nrf2-dependent regulation of ARE-mediated gene expression in Caco2 cells. In addition, the results provide experimental evidence for choosing the dose and frequency of the inducer in cancer chemoprevention study and in developing inhibitors of Nrf2-ARE signaling pathway.

Anticarcinogenic Agents ; pharmacology ; Antioxidants ; metabolism ; pharmacology ; Caco-2 Cells ; Calcium-Transporting ATPases ; antagonists & inhibitors ; Humans ; Hydroquinones ; pharmacology ; Isothiocyanates ; NF-E2-Related Factor 2 ; genetics ; metabolism ; physiology ; Oxidative Stress ; genetics ; physiology ; Response Elements ; physiology ; Signal Transduction ; drug effects ; Thiocyanates ; pharmacology

OBJECTIVETo investigate the effect of phenylhexyl isothiocyanate (PHI) on the drug-resistance to imatinib in K562/G01 cell line and to elucidate its possible mechanisms.

METHODSMTT assay was employed to access K562/G01 cell growth inhibition after exposure to PHI and/or imatinib at different concentrations. Apoptotic rate of K562/G01 cells was measured by flow cytometry. The levels of P-gp, P210(bcr-abl) and p-P210(bcr-abl) protein were detected by Western blot.

RESULTSPHI inhibited proliferation and induced apoptosis of K562/G01 cells after treated with PHI alone for 24 h. PHI concentration increased from 0 to 40 µmol/L, the inhibitory rate of cell proliferation from 0 to (51.22 ± 1.41)%, and the apoptosis rate from (3.76 ± 1.46)% to (35.35 ± 3.70)%. Combination of 10, 20, 40 µmol/L PHI and various concentrations of imatinib, IC50 s of imatinib were (10.49 ± 1.24), (6.33 ± 1.42), and (0.85 ± 0.17) µmol/L, respectively. When K562/G01 cells treated with 20 µmol/L PHI combined with 10 and 20µmol/L imatinib for 24 hours, apoptosis rate were (43.62 ± 4.23)% and (55.41 ± 4.35)%, respectively, being significantly higher than that with imatinib or PHI alone. PHI concentrations increased from 0 to 40 µmol/L for 7 hours, the P210(bcr-abl)/β-actin decreased from (0.944 ± 0.034) to (0.392 ± 0.025), and the p-P210(bcr-abl)/β-actin decreased from (0.906 ± 0.019) to (0.361 ± 0.021), while the alteration of P-gp was not seen.

CONCLUSIONSPHI inhibits the proliferation and induces apoptosis of K562/G01 cell line. PHI has synergistic effect with imatinib. It partially reverses the drug-resistance to imatinib. The mechanism of reversal of drug resistance in K562/G01 cells might be by inhibiting P210(bcr-abl) and p-P210(bcr-abl).

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Apoptosis ; drug effects ; Benzamides ; pharmacology ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; Isothiocyanates ; pharmacology ; K562 Cells ; Piperazines ; pharmacology ; Pyrimidines ; pharmacology