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1.
Apoptosis ; 22(11): 1362-1379, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28836036

ABSTRACT

Cancer cells often have faulty apoptotic pathways resulting in sustenance of survivability, tumour metastasis and resistance to anticancer drugs. Alternate strategies are sought to improve therapeutic efficacy and therefore HepG2 cells were treated with S-allyl-cysteine (SAC) and berberine (BER) to analyze their mechanistic impact upon necroptosis along with its interacting relationship to apoptosis. In the present study we observed that SAC and BER exposure reduced NFκß nuclear translocation through adenylate cyclase-cAMP-protein kinaseA axis and eventually evaded c-FLIP inhibition. Effective RIP1 k63-polyubiquitination and persistent MKK3/MKK6 expression during drug treatment potentiated caspase8 activity via p53-DISC conformation. Resultant tBid associated lysosomal protease mediated AIF truncation induced DNA fragmentation and persuaded effector caspase mediated scramblase activation resulting induction of necroptosis in parallel to apoptotic events. SAC+BER effectively reduced Rb-phosphorylation resulting insignificant nuclear E2F presence led to ending of cell proliferation. Therefore necroptosis augmented the drug response and may be targeted alongside cell proliferation inhibition in formation of efficient therapeutics against liver cancer.


Subject(s)
Adenylyl Cyclases/genetics , Berberine/pharmacology , Cysteine/analogs & derivatives , MAP Kinase Kinase 3/genetics , MAP Kinase Kinase 6/genetics , Signal Transduction , Adenylyl Cyclases/metabolism , Apoptosis/drug effects , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 8/genetics , Caspase 8/metabolism , Cell Line , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Cysteine/pharmacology , DNA Fragmentation/drug effects , Drug Combinations , Gene Expression Regulation , Hep G2 Cells , Humans , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase 6/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Necrosis/chemically induced , Necrosis/genetics , Necrosis/metabolism , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Protein Transport , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
2.
Biochim Biophys Acta ; 1840(1): 219-44, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23999088

ABSTRACT

BACKGROUND: Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN+CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated. METHODS: ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1-pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry. RESULTS: CytochromeP4502E1 mediated bioactivation of DEN+CCl4 induced Akt dependent pMdm2-HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2-HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure. CONCLUSION AND GENERAL SIGNIFICANCE: SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN+CCl4 induced hepatocarcinoma.


Subject(s)
Antineoplastic Agents/pharmacology , Berberine/pharmacology , Carbon Tetrachloride/toxicity , Carcinoma, Hepatocellular/prevention & control , Cysteine/analogs & derivatives , Diethylnitrosamine/toxicity , Liver Neoplasms/prevention & control , Alkylating Agents/toxicity , Animals , Apoptosis/drug effects , Blotting, Western , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/metabolism , Caspase 3/metabolism , Cells, Cultured , Cysteine/pharmacology , Cytochrome P-450 CYP2E1/metabolism , Cytochromes c/metabolism , Flow Cytometry , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Histone Deacetylase 1/metabolism , Immunoenzyme Techniques , Immunoprecipitation , Liver Neoplasms/chemically induced , Liver Neoplasms/metabolism , Male , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism
3.
Biochim Biophys Acta ; 1820(1): 9-23, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22033380

ABSTRACT

BACKGROUND: Chronic lead (Pb(2+)) exposure leads to the reduced lifespan of erythrocytes. Oxidative stress and K(+) loss accelerate Fas translocation into lipid raft microdomains inducing Fas mediated death signaling in these erythrocytes. Pathophysiological-based therapeutic strategies to combat against erythrocyte death were evaluated using garlic-derived organosulfur compounds like diallyl disulfide (DADS), S allyl cysteine (SAC) and imidazole based Gardos channel inhibitor clotrimazole (CLT). METHODS: Morphological alterations in erythrocytes were evaluated using scanning electron microscopy. Events associated with erythrocyte death were evaluated using radio labeled probes, flow cytometry and activity gel assay. Mass spectrometry was used for detection of GSH-4-hydroxy-trans-2-nonenal (HNE) adducts. Fas redistribution into the lipid rafts was studied using immunoblotting technique and confocal microscopy. RESULTS: Combination of SAC and CLT was better than DADS and CLT combination and monotherapy with these agents in prolonging the survival of erythrocytes during chronic Pb(2+) exposure. Combination therapy with SAC and CLT prevented redistribution of Fas into the lipid rafts of the plasma membrane and downregulated Fas-dependent death events in erythrocytes of mice exposed to Pb(2+). CONCLUSION AND GENERAL SIGNIFICANCE: Ceramide generation was a critical component of Fas receptor-induced apoptosis, since inhibition of acid sphingomyelinase (aSMase) interfered with Fas-induced apoptosis during Pb(2+) exposure. Combination therapy with SAC and CLT downregulated apoptotic events in erythrocytes by antagonizing oxidative stress and Gardos channel that led to suppression of ceramide-initiated Fas aggregation in lipid rafts. Hence, combination therapy with SAC and CLT may be a potential therapeutic option for enhancing the lifespan of erythrocytes during Pb(2+) toxicity.


Subject(s)
Clotrimazole/pharmacology , Cysteine/analogs & derivatives , Erythrocytes/drug effects , Lead Poisoning/blood , fas Receptor/metabolism , Animals , Apoptosis , Cysteine/pharmacology , Down-Regulation/drug effects , Erythrocytes/metabolism , Erythrocytes/pathology , Female , Lead/toxicity , Lead Poisoning/pathology , Mass Spectrometry , Mice , Mice, Inbred BALB C , Reactive Oxygen Species , Signal Transduction
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