Tracking anti‐fibrotic pathways of nilotinib and imatinib in experimentally induced liver fibrosis: An insight

The tyrosine kinase inhibitors imatinib and nilotinib have been suggested to have promising antifibrotic activity in experimental models of liver fibrosis. The aim of the present study was to investigate new pathways underlying this beneficial effect. Hepatic injury was induced in male Wistar rats by intraperitoneal injection of CCl₄ for 12 weeks. During the last 8 weeks of treatment, rats were also injected daily intraperitoneally with 20 mg/kg imatinib or 20, 10 or 5 mg/kg nilotinib. At the end of treatment, effects on fibrosis were assessed by measuring serum fibrotic markers and profibrogenic cytokines, as well as by histopathological examination. Possible anti‐inflammatory effects were estimated by measuring levels of inflammatory cytokines in liver tissue. Liver expression of α‐smooth muscle actin, transforming growth factor (TGF)‐β1 antibodies and platelet‐derived growth factor receptor β (PDGFRβ) was evaluated by immunohistochemical staining techniques. Nilotinib (5 and 10 mg/kg) significantly (P < 0.05) decreased all serum fibrotic markers measured, but 20 mg/kg of either nilotinib or imatinib had limited effects. At all doses tested, nilotinib significantly (P < 0.05) decreased the CCl₄‐induced increases in tissue inflammatory cytokines. Furthermore, 5 and 10 mg/kg nilotinib significantly decreased TGF‐β1 levels and tissue expression of its antibody, as well expression of PDGFRβ. In conclusion, low doses (5 and 10 but not 20 mg/kg) of nilotinib, rather than imatinib, can control hepatic fibrosis by regulating levels of proinflammatory cytokines, primarily interleukin (IL)‐1 and IL‐6. Nilotinib also controls the signalling pathways of profibrogenic cytokines by lowering TGF‐β1 levels and decreasing expression of PDGFRβ.     

Antifibrotic effects of D‐limonene (5(1‐methyl‐4‐[1‐methylethenyl]) cyclohexane) in CCl4 induced liver toxicity in Wistar rats

This study was designed to assess the potential antifibrotic effect of D‐Limonene—a component of volatile oils extracted from citrus plants. D‐limonene is reported to have numerous therapeutic properties. CCl₄‐intduced model of liver fibrosis in Wistar rats is most widely used model to study chemopreventive studies. CCl₄‐intoxication significantly increased serum aminotransferases and total cholesterol these effects were prevented by cotreatment with D‐Limonene. Also, CCl₄‐intoxication caused depletion of glutathione and other antioxidant enzymes while D‐Limonene preserved them within normal values. Hydroxyproline and malondialdehyde content was increased markedly by CCl₄ treatment while D‐Limonene prevented these alterations. Levels of TNF‐α, TGF‐β, and α‐SMA were also assessed; CCl₄ increased the expression of α‐SMA, NF‐κB and other downstream inflammatory cascade while D‐Limonene co‐treatment inhibited them. Collectively these findings indicate that D‐Limonene possesses potent antifibrotic effect which may be attributed to its antioxidant and anti‐inflammatory properties.     

Aflatoxin B1 affects apoptosis and expression of Bax,Bcl‐2, and Caspase‐3 in thymus and bursa of fabricius in broiler chickens

Aflatoxin B₁ is known as a mycotoxin that develops various health problems of animals, the effects of AFB₁ on thymus and bursa of Fabricius in chickens are not clear. The objective of this study was to investigate the apoptosis of thymus and bursa of Fabricius in broilers fed with AFB₁. Two hundred Avian broilers were randomly divided into four groups of 50 each, namely control group and three AFB₁ groups fed with 0.15 mg, 0.3 mg, and 0.6 mg AFB₁/kg diet, respectively. In this study, flow cytometer and immunohistochemical approaches were used to determine the percentage of apoptotic cells and the expression of Bax, Bcl‐2, and Caspase‐3. The results showed that consumption of AFB₁ diets results in increased percentage of apoptotic cells and increased expression of Caspase‐3 in both thymus and bursa of Fabricius. The expression of Bax was increased and the expression of Bcl‐2 was decreased in the thymus, but no significant changes in Bax and Bcl‐2 expression were observed in the bursa of Fabricius when broilers fed with AFB₁. These findings suggest that adverse effects of AFB₁ on thymus and bursa of Fabricius in broilers were confirmed by increased apoptotic cells and abnormal expression of Caspase‐3. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1113–1120, 2016.     

Betulinic acid prevention of d-galactosamine/lipopolysaccharide liver toxicity is triggered by activation of Bcl-2 and antioxidant mechanisms

Objectives The hepatoprotective activity and molecular mechanism of betulinic acid (BA) was investigated on acute liver failure induced by d ‐galactosamine (D‐GalN)/lipopolysaccharide (LPS) in vivo. Methods Mice were administered with different doses of BA (20 mg/kg or 50 mg/kg, i.p.) 1 h before injection of D‐GalN (700 mg/kg)/LPS (10 µg/kg) and sacrificed 6 h after treatment with D‐GalN/LPS. Key findings Pretreatment with BA significantly prevented the increases of serum aspartate aminotransferase and alanine aminotransferase, while it increased the content of glutathione and catalase, and reduced malondialdehyde. BA showed obvious anti‐oxidant effects and prevented D‐GalN/LPS‐induced apoptosis, as indicated by DNA ladder. BA treatment resulted in regulation of the mitogen‐activated protein kinase. We found that BA mediated production of c‐jun NH₂‐terminal protein kinase and extracellular signal‐regulated kinase induced by D‐GalN/LPS, promoted the expression of B‐cell CLL/lymphoma 2 (Bcl‐2) and restored mitochondrial outer membrane permeabilization. Conclusions The results suggested that BA prevented D‐GalN/LPS‐induced acute liver failure by upregulation of Bcl‐2 and antioxidation and mediation of cytokines causing apoptotic cell death and lessened liver damage.     

AlCl3 induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway

To investigate apoptosis mechanisms in lymphocytes induced by aluminum trichloride (AlCl₃) through the mitochondria–caspase dependent pathway, the spleen lymphocytes of rats were cultured with RPMI‐1640 medium and exposed to AlCl₃·6H₂O in the final concentrations of 0 (control group, CG), 0.3 (low‐dose group, LG), 0.6 (mid‐dose group, MG), and 1.2 (high‐dose group, HG) mmol·L^?1 for 24 h, respectively. Mitochondrial transmembrane potential (ΔΨm), cytochrome C (Cyt C) protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, DNA ladder and lymphocytes apoptosis index were detected. The results showed that Cyt C protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, the ratio of Bcl‐2 and Bax mRNA expression, lymphocytes apoptosis index increased, while ΔΨm decreased in the AlCl₃‐treated groups compared with those in CG. The results indicate that AlCl₃ induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 385–394, 2016.     

Naringenin attenuates CCl4‐induced hepatic inflammation by the activation of an Nrf2‐mediated pathway in rats

The possible protective effects of naringenin, a naturally occurring citrus flavonone, on carbon tetrachloride (CCl₄)‐induced liver injury in rats and the mechanism underlying its effects were investigated. Forty rats were divided into five groups. Rats in Groups I and II served as the normal and injured liver groups, respectively; Group III rats were treated with the standard drug silymarin as a positive control; and rats in Groups IV and V (naringenin‐treated groups) were administrated 50 mg/kg, p.o., naringenin for 7 days. Liver samples were collected to evaluate mRNA and protein expression, histological changes and oxidative stress. Naringenin inhibited lipid peroxidation and reduced serum levels of hepatic enzymes induced by CCl₄. In addition, naringenin increased the liver content of reduced glutathione and the activity of anti‐oxidant enzymes in rats treated with CCl₄. Naringenin attenuated liver inflammation by downregulating CCl₄‐induced activation of tumour necrosis factor (TNF)‐α, inducible nitric oxide synthase (iNOS) and cyclo‐oxygenase (COX‐2) at both the protein and mRNA levels. Naringenin treatment significantly increased NF‐E2‐related factor 2 (Nrf2) and heme oxygenase (HO‐1) expression in injured livers. In rats treated with CCl₄ alone, decreases were seen in nuclear Nrf2 expression and in the mRNA levels of its target genes (e.g. HO‐1, NQO1 and glutathione S‐transferase alpha 3 (GST‐a3)). Together, the results suggest that naringenin can protect the liver against oxidative stress, presumably by activating the nuclear translocation of Nrf2 as well as attenuating the TNF‐α pathway to elicit an anti‐inflammatory response in liver tissue.     

Deferoxamine alleviates liver fibrosis induced by CCl4 in rats

Several chronic liver diseases can lead to the occurrence of hepatic fibrosis through the accumulation of iron, which causes induction of oxidative stress and consequently activation of fibrogenesis. The present study was designed to investigate the potential antifibrotic and anti‐oxidant effects of deferoxamine (DFO), a well‐known iron chelator in an experimental rat model of liver injury using carbon tetrachloride (CCl₄). First, the potential effective dose of DFO was screened against CCl₄‐induced acute hepatotoxicity. Then, rats were co‐treated with DFO (300 mg/kg, i.p.) for 6 weeks starting from the third week of CCl₄ induction of chronic hepatotoxicity. Liver function was assessed in addition to histopathological examination. Furthermore, oxidative stress and fibrosis markers were assessed. It was found that treatment of animals with DFO significantly counteracted the changes in liver function; histopathological lesions and hepatic iron deposition that were induced by CCl₄. DFO also significantly counteracted the CCl₄‐induced lipid peroxidation increase and reduction in antioxidant activities of superoxide dismutase and glutathione peroxidase enzymes. In addition, DFO ameliorated significantly liver fibrosis markers including hydroxyproline, collagen accumulation, and the expression of the hepatic stellate cells (HSCs) activation marker; alpha smooth muscle actin and transforming growth factor‐beta (TGF‐β). Together, these findings indicate that DFO possesses a potent antifibrotic effect due to its antioxidant properties that counteracted oxidative stress and lipid peroxidation and restored antioxidant enzymes activities as well as reducing HSCs activation and fibrogenesis.     

TGF‐β1 mediated Smad signaling pathway and EMT in hepatic fibrosis induced by Nano NiO in vivo and in vitro

Nickel oxide nanoparticles (Nano NiO) bears hepatotoxicity, while whether it leads to liver fibrosis remains unclear. The aim of this study was to establish the Nano NiO‐induced hepatic fibrosis model in vivo and investigate the roles of transforming growth factor β1 (TGF‐β1) in Smad pathway activation, epithelial‐mesenchymal transition (EMT) occurrence, and extracellular matrix (ECM) deposition in vitro. Male Wistar rats were exposed to 0.015, 0.06, and 0.24 mg/kg Nano NiO by intratracheal instilling twice a week for 9 weeks. HepG2 cells were treated with 100 μg/mL Nano NiO and TGF‐β1 inhibitor (SB431542) to explore the mechanism of collagen formation. Results of Masson staining as well as the elevated levels of type I collagen (Col‐I) and Col‐III suggested that Nano NiO resulted in hepatic fibrosis in rats. Furthermore, Nano NiO increased the protein expression of TGF‐β1, p‐Smad2, p‐Smad3, alpha‐smooth muscle actin (α‐SMA), matrix metalloproteinase9 (MMP9), and tissue inhibitors of metalloproteinase1 (TIMP1), while decreased the protein content of E‐cadherin and Smad7 in rat liver and HepG2 cells. Most importantly, Nano NiO‐triggered the abnormal expression of the abovementioned proteins were all alleviated by co‐treatment with SB431542, implying that TGF‐β1‐mediated Smad pathway, EMT and MMP9/TIMP1 imbalance were involved in overproduction of collagen in HepG2 cells. In conclusion, these findings indicated that Nano NiO induced hepatic fibrosis via TGF‐β1‐mediated Smad pathway activation, EMT occurrence, and ECM deposition.     

Antifibrotic effect of AT‐1 blocker and statin in rats with hepatic fibrosis

Hepatic fibrosis is an outcome of chronic liver injury. Angiotensin II (ANG II) may play a role in the pathogenesis of hepatic fibrosis. Certain drugs such as ACE inhibitors, ANG II antagonists, and even statins could interfere with the renin angiotensin system and modulate its deleterious effects. This study was carried out to investigate the possible role of losartan and atorvastatin in liver fibrosis. Liver fibrosis was induced in rats by i.p. injection of 50% CCl₄ twice per week for 8 weeks. The rats intoxicated with CCl₄ were divided into four groups: fibrosis control; losartan group; atorvastatin group; and co‐treated group. A fifth group of normal healthy rats served as a control group. The results showed that losartan and atorvastatin, either alone or in combination, significantly decreased ALT, AST, hyaluronic acid and hydroxyproline levels in their groups compared to those of the fibrosis control group. A significant decrease in TGF‐β was found in the losartan and co‐treated groups but not in the atorvastatin group. These biochemical data were supported by liver histopathology and α‐SMA. The results indicate that the combined treatment with both losartan and atorvastatin produced a greater effect than either drug alone and proved a beneficial role in inhibiting or reversing liver fibrosis.     

Increasing the effectiveness of tyrosine kinase inhibitor (TKI) in combination with a statin in reducing liver fibrosis

It has been shown that both nilotinib as a tyrosine kinase inhibitor, and atorvastatin as a rho‐kinase inhibitor, have antifibrotic effects. Therefore, considering the relationship between these two pathways, this study aimed to investigate the effects of their co‐treatment against hepatic stellate cells (HSCs) activation and liver fibrosis. For this purpose, the activation of HSCs coincided with these therapies. Also, liver fibrosis by carbon tetrachloride (CCl₄) was induced in male Wistar rats and treated simultaneously with these compounds. The expression of alpha‐smooth muscle actin (α‐SMA), connective tissue growth factor (CTGF), Ras homolog gene family, and member A (RhoA)/Rho‐associated protein kinase (ROCK) in HSCs were measured. The expression of transforming growth factor beta‐1 (TGF‐β1), its receptor (TβRII), CTGF, and platelets derived growth factor (PDGF), in the livers, were also investigated, all by real‐time PCR and western blot analysis. Also, histopathologic and immunohistochemical evaluations were performed to evaluate changes in liver fibrosis during treatment. The results indicated the down‐regulation of RhoA/ROCK, CTGF, and α‐SMA, and inhibition of the HSCs activation toward myofibroblasts. The results also showed that the combined use of atorvastatin and nilotinib has significantly higher inhibitory effects. The antifibrotic effects of atorvastatin and nilotinib co‐administration were also observed by histopathologic and immunohistochemical observations, and inhibiting the expression of TGF‐β1, TβRII, CTGF, and PDGF. Taken together, this study revealed that co‐administration of nilotinib–atorvastatin has novel antifibrotic effects, by inhibiting RhoA/ROCK, and CTGF pathway. Therefore, the importance of the common pathway of RhoA/ROCK and CTGF, in reducing fibrosis may almost be concluded.     

Role of Bcl‐2 in tumour cell survival and implications for pharmacotherapy

Objectives Bcl‐2 is a protein that inhibits apoptosis, leading to cell survival. The Bcl‐2 family has six different anti‐apoptotic proteins, three pro‐apoptotic proteins that are similar in structure, and other integrating proteins that function as promotors or inhibitors in the progression of apoptosis. In this discussion paper, we provide an overview of apoptosis, the role of Bcl‐2 in normal cellular and molecular processes, and the role of Bcl‐2 in tumour cell survival. It focuses primarily on anti‐apoptotic Bcl‐2, its activation in cancer, the manner in which it regulates the intrinsic and extrinsic mechanisms of apoptosis, and its broad molecular interactions with other critical proteins in the cell. Certain cancer treatments are reviewed and related directions for the future are presented. Key findings Apoptosis is common to all organisms – for eukaryotes it is a normal process of development and regeneration. The rate at which apoptosis occurs is critical to the survival of the organism, as too much can lead to the onset of degenerative diseases such as dementia, and too little may lead to cancer. FKBP‐38 is a binding protein that has been discovered to be upregulated in highly aggressive cancers and binds to Bcl‐2 rather than the pro‐apoptotics to induce a state of hyper‐mitosis. A short binding protein (Nur‐77) provides new insights into Bcl‐2 ‘masking’. Nurr‐77 binds to Bcl‐2 and exposes the BH3 domain, transforming it from a cancer promoter to an unorthodox cancer inhibitor. This presents in itself an interesting and exciting opportunity – increasing the rate of apoptosis in neoplastic cells that are usually protected by Bcl‐2 activity at the mitochondria. Summary Development of drugs in the form of BH3‐only and BH123 mimetic drugs provide a interesting avenue for cancer therapy for the future. Drugs that can either promote, or mimic anti‐IAP activity such as Smac/Diablo would certainly be productive, thereby inducing apoptosis. Medicinal usage which can effectively suppress FKBP38 in Bcl‐2‐dependent cancers would provide further arsenal to combat apoptotic irregularities, particularly a treatment that is more dominant than kinetin riboside. WAVE‐1 inhibitors may effectively suppress the phosphorylation of Bcl‐2, thereby potentially reducing hyper‐mitosis and increasing apoptosis. Recent findings shed molecular light on PDT, namely ER stress, and potential for anti‐cancer therapy via either apoptosis or autophagy. A drug that can effectively upregulate Nurr‐77, thereby masking the anti‐apoptotic properties of Bcl‐2, would indeed be life‐saving for cancer patients.     

Arylazopyrazole AAP1742 Inhibits CDKs and Induces Apoptosis in Multiple Myeloma Cells via Mcl‐1 Downregulation

Inhibitors of cyclin‐dependent kinases 9 have been developed as potential anticancer drugs for the treatment of multiple myeloma. We have previously prepared a library of arylazo‐3,5‐diaminopyrazole inhibitors of CDKs. Here, we describe a novel member, AAP1742 (CDK9 inhibition with IC₅₀ = 0.28 μm ), that reduces the viability of multiple myeloma cell lines in low micromolar concentrations. Consistent with inhibition of CDK9, AAP1742 decreases the phosphorylation of RNA polymerase II and inhibits mRNA synthesis of anti‐apoptotic proteins Mcl‐1, Bcl‐2, and XIAP, followed by apoptosis in the RPMI‐8226 cell line in a dose‐ and a time‐dependent manner. These results are consistent with the biochemical profile of AAP1742 and further suggest cellular inhibition of CDK9 as a possible target for anticancer drugs.     

Inhibition of TGFβ type I receptor activity facilitates liver regeneration upon acute CCl4 intoxication in mice

Liver exhibits a remarkable maintenance of functional homeostasis in the presence of a variety of damaging toxic factors. Tissue regeneration involves cell replenishment and extracellular matrix remodeling. Key regulator of homeostasis is the transforming growth factor-β (TGFβ) cytokine. To understand the role of TGFβ during liver regeneration, we used the single-dose carbon tetrachloride (CCl₄) treatment in mice as a model of acute liver damage. We combined this with in vivo inhibition of the TGFβ pathway by a small molecule inhibitor, LY364947, which targets the TGFβ type I receptor kinase [activin receptor-like kinase 5 (ALK5)] in hepatocytes but not in activated stellate cells. Co-administration of LY364947 inhibitor and CCl₄ toxic agent resulted in enhanced liver regeneration; cell proliferation (measured by PCNA, phosphorylated histone 3, p21) levels were increased in CCl₄ + LY364947 versus CCl₄-treated mice. Recovery of CCl₄-metabolizing enzyme CYP2E1 expression in hepatocytes is enhanced 7 days after CCl₄ intoxication in the mice that received also the TGFβ inhibitor. In summary, a small molecule inhibitor that blocks ALK5 downstream signaling and halts the cytostatic role of TGFβ pathway results in increased cell regeneration and improved liver function during acute liver damage. Thus, in vivo ALK5 modulation offers insight into the role of TGFβ, not only in matrix remodeling and fibrosis, but also in cell regeneration.     

Potential protective effects of a traditional Chinese herb,Litsea coreana Levl., on liver fibrosis in rats

Objectives It was found that total flavonoids from Litsea coreana Levl. (TFLC), which is a traditional Chinese medicine, had a preventive effect against hepatic steatosis in our previous study. This study was designed to evaluate whether TFLC could improve liver fibrosis in rats. Methods The liver fibrosis model rats were treated with composite factors of high‐fat emulsion (10 ml/kg) via gavage accompanied by a subcutaneous injection of low‐dose CCl₄. Thirty rats were given composite factors plus TFLC (100, 200, 400 mg/kg), respectively, for 8 weeks. Key findings The results showed that TFLC (200 and 400 mg/kg) treatment significantly reduced the elevation of liver index (liver weight/body weight) and spleen index (spleen weight/body weight), alanine transaminase, aspartate aminotransferase, hyaluronic acid, laminin, procollagen III N‐terminal peptide, procollagenase IV and hydroxyproline. In addition, TFLC treatment improved the morphologic changes of hepatic fibrosis, suppressed expression of α‐smooth muscle actin, collagen I, transforming growth factor (TGF)‐β1 and TGFβ receptor (TGFβR)1, and increased peroxisome proliferator‐activated receptor‐γ expression in the liver of hepatic fibrosis rats. Conclusions In conclusion, TFLC is able to ameliorate liver injury and protect rats from liver fibrosis. This process may be related to inhibiting the expression of transforming growth factor‐β1 and increasing the expression of peroxisome proliferator‐activated receptor‐γ.     

Toxicity assessment of manganese oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral exposure

In the near future, nanotechnology is envisaged for large‐scale use. Hence health and safety issues of nanoparticles (NPs) should be promptly addressed. Twenty‐eight‐day oral toxicity, genotoxicity, biochemical alterations, histopathological changes and tissue distribution of nano and microparticles (MPs) of manganese oxide (MnO₂) in Wistar rats was studied. Genotoxicity was assessed using comet, micronucleus and chromosomal aberration assays. The results demonstrated a significant increase in DNA damage in leukocytes, micronuclei and chromosomal aberrations in bone marrow cells after exposure of MnO₂‐NPs at 1000, 300 mg kg^–1 bw per day and MnO₂‐MPs at the dose of 1000 mg kg^–1 bw per day. Our findings showed acetylcholinestrase inhibition at 1000 as well as at 300 mg kg^–1 bw per day in blood and with all the doses in the brain indicating the toxicity of MnO₂‐NPs. Further, the doses significantly inhibited different ATPases in the brain P₂ fraction. Significant changes were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in the liver, kidney and serum in a dose‐dependent manner. MnO₂‐MPs at 1000 mg kg^–1 bw per day were found to induce significant alterations in biochemical enzymes. A significant distribution was found in all the tissues in a dose‐dependent manner. MnO₂‐NPs showed a much higher absorptivity and tissue distribution as compared with MnO₂‐MPs. A large fraction of MnO₂‐NPs and MnO₂‐MPs was cleared by urine and feces. Histopathological analysis revealed that MnO₂‐NPs caused alterations in liver, spleen, kidney and brain. The MnO₂‐NPs induced toxicity at lower doses compared with MnO₂‐MPs. Further, this study did not display gender differences after exposure to MnO₂‐NPs and MnO₂‐MPs. Therefore, the results suggested that prolonged exposure to MnO₂ has the potential to cause genetic damage, biochemical alterations and histological changes. Copyright © 2013 John Wiley & Sons, Ltd.     

Lanthanum chloride promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes

Population surveys and animal experiments have shown that rare earth elements (REEs) cause neurological defects. However, the detailed mechanisms underlying these effects are still unclear. Given that lanthanum is commonly used for investigating into REEs‐induced neurological defects, this study chose lanthanum chloride (LaCl₃) to show that LaCl₃ promotes mitochondrial apoptotic pathway in primary cultured rat astrocytes by regulating expression of Bcl‐2 family proteins. The main findings of this study are (1) LaCl₃ treatment (0.25, 0.5, and 1.0 mM for 12–48 h) induced the astrocytes damages with a concentration‐dependent manner, which were confirmed with methyl thiazolyl tetrazolium and lactate dehydrogenase release assays, and morphological examination. (2) A 24 h treatment of LaCl₃ concentration‐dependently decreased mitochondrial membrane potential, increased cytochrome c release from mitochondria into cytosol, elevated caspase 9 and 3 expression, and promoted astrocyte apoptosis. (3) LaCl₃ treatment increased the ratio of pro‐apoptotic Bax and antiapoptotic Bcl‐2 proteins, which in turn broke the balance among pro‐apoptotic and antiapoptotic Bcl‐2 family proteins, leading to astrocyte apoptosis. Our results indicate that LaCl₃ alters Bcl‐2 family protein expressions, which in turn promote mitochondrial apoptotic pathway, and thus astrocytic damage. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 489–497, 2013.     

Effect of HA14‐1 on Apoptosis‐Regulating Proteins in HeLa Cells

Overexpression of Bcl‐2 has been recognized in various malignancies. Recently, HA14‐1, a Bcl‐2 antagonist, has been identified for its anti‐apoptotic effect. However, mode of action of HA14‐1 still remains to be elucidated. In this study, we examined HA14‐1 binding efficiency with receptor proteins through molecular docking. Cell viability using HeLa cells was evaluated through MTT assay after exposure to different concentration of HA14‐1. Moreover, after HA14‐1 exposure, expressions of tumor suppressor protein (p53), BH3‐only protein (Puma) and apoptosis‐associated proteins were analyzed by Western blotting. From the results, it was found that HA14‐1 occupied all three domains; BH1, BH2, and BH3 within the hydrophobic pocket of Bcl‐2. However, HA14‐1 occupied only BH1 and BH3 of Bcl‐xl, conversely, no such stable bond was observed for Bax and Bak. ARG107 and TYR101 were the amino acids involved in the binding of HA14‐1 to Bcl‐2 and Bcl‐xl, respectively. Additionally, decrease in Bcl‐2 and Bcl‐xl expression along with increase in p53 and Puma expression after exposure to HA14‐1 was observed. The results suggested p53 pathway to be the probable mechanism of action for the induction of apoptosis in HeLa cell by downregulating the effect of anti‐apoptotic proteins suggesting that HA14‐1 may provide therapeutic potential for the treatment of human cervical cancer.