Perfluorooctanoic acid disrupts gap junction intercellular communication and induces reactive oxygen species formation and apoptosis in mouse ovaries

Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acid family of compounds. Due to the presence of strong carbon–fluorine bonds, it is practically nonbiodegradable and highly persistent in the environment. PFOA has been detected in the follicular fluid of women, and positively associated with reduced fecundability and infertility. However, there are no reports concerning the experimental evaluation of PFOA on oocyte toxicity in mammals. The aim of the present study was to determine if PFOA is able to induce oxidative stress in fetal ovaries and cause apoptosis in oocytes in vitro. In addition, since inhibition of the gap junction intercellular communication (GJIC) by PFOA has been demonstrated in liver cells in vivo and in vitro, the effect of PFOA on the GJIC between the oocyte and its supportive cumulus cells was studied. Results show that PFOA induced oocyte apoptosis and necrosis in vitro (medium lethal concentration, LC₅₀ = 112.8 μM), as evaluated with Annexin‐V‐Alexa 508 in combination with BOBO‐1 staining. Reactive oxygen species (ROS) levels, as assessed by DCFH‐DA, increased significantly in fetal ovaries exposed to ¼ LC₅₀ (28.2 μM, a noncytotoxic and relevant occupational exposure concentration) and LC₅₀ PFOA ex vivo. This perfluorinated compound also caused the blockage of GJIC in cumulus cells‐oocyte complexes (COCs) obtained from female mice exposed in vivo, as evaluated by calcein transfer from cumulus cells to the oocyte. The ability of PFOA of disrupting the GJIC in COCs, generating ROS in the fetal ovary and causing apoptosis and necrosis in mammal's oocytes, might account for the reported association between increasing maternal plasma concentrations of PFOA with reduced fertility in women.     

Bystander effects of PC12 cells treated with Pb depend on ROS-mitochondria-dependent apoptotic signaling via gap-junctional intercellular communication

The demonstration of bystander effect, which means injured cells propagate damage to neighboring cells, in whole organisms has clear implication of the potential relevance of the non-targeted response to human health. Here we show that 10 μM lead acetate, the optimum concentration for inducing apoptosis confirmed by the expression levels of Bax and Bcl-2, can also induce rat pheochromocytoma (PC12) cells to exert bystander effects to neighboring cells. In a novel co-culture system, GFP-PC12 (Pb²⁺) cells, which were stable transfected with EF1A-eGFP and pre-exposed with lead acetate, were co-cultured with unexposed PC12 cells at a 1:5 ratio. Parachute assays demonstrated the functional gap-junctional intercellular communication (GJIC) formed between Pb²⁺-exposed and unexposed cells. The Pb²⁺-exposed cells induced very similar effects on neighboring unexposed cells to apoptosis coincide with intracellular ROS generation and the collapse of mitochondrial membrane potential (Δψm). Furthermore, carbenoxolone (CBX), a blocker of GJIC, inhibited the bystander effects. The results indicate that the Pb²⁺-induced insults propagate through GJIC between PC12 cells, while inducing the bystander cells to apoptosis via ROS-mitochondria-dependent apoptotic signaling.     

The protective effects of pretreatment with resveratrol in cyclophosphamide-induced rat ovarian granulosa cell injury: In vitro study

Cyclophosphamide (Cy), a chemotherapeutic agent, is widely used to treat tumoursand is also associated with premature ovarian insufficiency. 4-Hydroperoxycyclophosphamide (4-HC), an active metabolite of Cy, was used for in vitro experiments. Granulosa cells (GCs) are crucial for maintaining follicle development and are also used in reproductive toxicity research in vitro. Resveratrol (Res), a polyphenolic compound, exhibits multiple effects in cells and animal models. To date, whether Res pretreatment has a protective effect on GCs induced by Cy remains unclear. This was an in vitro study, and primary cultures of rat GCs were used. Rat GCs were treated with 4-HC alone, Res + 4-HC or Res + 4-HC + EX527, and GCs survival rates, oxidative stress levels, apoptosis rates and related Sirt1 pathway proteins were evaluated. We demonstrated that 4-HC caused GC damage by increasing oxidative stress, autophagy and apoptosis. Res pretreatment improved 4-HC-induced GC damage by increasing Sirt1 expression, reducing oxidative stress levels and decreasing Beclin1, LC3B, Bax and Caspase-3 levels. Importantly, the addition of EX527, which is a selective inhibitor of Sirt1, reversed the protective effect of Res pretreatment, indicating that Sirt1 may be an important mediator of the protective effect of Res. Taken together, we demonstrated that Res may be a potential drug to improve fertility preservation for patients undergoing chemotherapy.     

岩大戟内酯B对人卵巢癌Skov3细胞的增殖抑制作用

目的探讨岩大戟内酯B(JB)对人卵巢癌Skov3细胞的增殖抑制作用及可能机制。方法采用不同浓度的JB处理卵巢癌Skov3细胞48 h,采用四甲基偶氮唑盐(MTT)法检测Skov3细胞的增殖抑制率,Hoechst法检测Skov3细胞凋亡指数,Western blot检测Akt、P-Akt(Ser473)、Bax、Caspase-3和Bcl-2蛋白水平。结果 0.1,1.0,5.0,10.0,20.0,50.0μmol/L的JB可呈剂量依赖的方式抑制Skov3细胞的增殖,IC50为5.28μmol/L;20.0μmol/L JB和30.0μmol/L LY294002(PI3K抑制剂)均可增加Skov3细胞凋亡指数,降低P-Akt(Ser473)/Akt水平,增加促凋亡基因(Bax、Caspase-3)的蛋白水平,降低抑制凋亡基因(Bcl-2)的蛋白水平,且均与对照组有显著差异;而LY294002可增强JB的效应,且与JB组有显著差异。结论 JB可抑制人卵巢癌Skov3细胞的增殖,促进细胞凋亡,可能是通过抑制PI3K/Akt信号通路和诱导死亡受体表达来发挥抗肿瘤作用的。     

Carbenoxolone induces apoptosis and inhibits survivin and survivin-ΔEx3 genes expression in human leukemia K562 cells

Background and the purpose of the study

Leukemia is a malignant disorder of the blood progenitor/stem cells which is characterized by abnormal proliferation of white blood cells. Although anti-cancer drugs induce apoptosis in cancerous cells, drug resistance is the significant problem mainly due to over-expression of inhibitors of apoptosis proteins (IAPs) such as survivin. In this content, it has been reported that an anti-inflammatory drug, Carbenoxolone (CBX), could induce apoptosis and growth inhibition in several types of cancerous cells. In the present study, effects of CBX on apoptosis and level of the expression of survivin gene and its ΔEx3 splicing variant have were evaluated in K562 cells.

Methods

K562 cells were cultured and treated with different concentrations of CBX (50-300 µM) at different time intervals (12-48 hrs). Trypan blue exclusion test was used to evaluate cell viability. Fluorescent microscopy (Acridine Orange/Ethidium Bromide double staining) and DNA fragmentation assay were used to study apoptosis. The expression level of survivin and its ΔEx3 splice variant were studied by RT-PCR.

Results and Major Conclusion

It was found that both growth inhibition and apoptosis occurred in K562 cells. In addition, down-regulation of survivin and survin-ΔEx3 were observed, after 2-4 hrs treatment with 150 µM of CBX. However, the expression level of survivin and its ΔEx3 splice variant increased in subsequent time (6-12 hrs) nearly to the level of control cells. From the results of this study, it may be concluded that CBX can be considered as a candidate for further studies in CML treatment, especially in the case of drug-resistant leukemia cells.     

二氯乙酸钠联合顺铂诱导人肺腺癌A549细胞株凋亡的实验研究

目的研究二氯乙酸钠(Sodium dichloroacetate,DCA-Na)、顺铂(DDP)联合对人肺腺癌A549细胞株增殖和凋亡的影响及其作用机制。方法用MTT法检测DCA-Na、DDP应用对A549细胞增殖抑制作用的影响;流式细胞仪(Annexin V-FITC/PI法)检测DCA-Na、DDP单药及两药联合作用于A549细胞凋亡率的变化;用分光光度法检测DCA-Na作用于A549细胞后半胱氨酸天门冬氨酸蛋白酶(Caspase)-3、-8、-9蛋白的活性。结果DCA-Na、DDP单药组均对A549细胞增殖有抑制作用,且呈明显的剂量-时间依赖性。0.4、2μg/mL的DDP与37.5、75、150μg/mL的DCA-Na联合作用A549细胞24、48、72 h后的抑制率显著高于同浓度DDP单药组(P<0.05),且2μg/mL的DDP与75μg/mL的DCA联合在48 h表现为协同作用。流式细胞仪检测显示,A549细胞联合组凋亡率显著高于各单药组(P<0.05)。DCA-Na作用于A549细胞后,分别在12、24、48、72 h测得Caspase-3、-8、-9蛋白活性显著高于对照组(P<0.05)。结论 DCA-Na对人肺腺癌A549细胞的增殖有抑制作用,并且随着药物浓度增加、作用时间延长,其抑制作用也增加(呈剂量和时间依赖性)。一定浓度范围的DCA-Na和DDP联合作用于人肺腺癌A549细胞能够产生协同作用。DCA-Na可诱导人肺腺癌A549细胞凋亡,且与DDP联合后其诱导凋亡作用更为显著。     

Synergistic interaction between trastuzumab and EGFR/HER-2 tyrosine kinase inhibitors in HER-2 positive breast cancer cells

Overexpression of HER-2 in breast cancer is frequently associated with expression of EGFR, and EGFR expression influences response to HER-2 inhibition. The aim of this study was to examine the effects of combining dual inhibition of EGFR and HER-2, using trastuzumab, gefitinib and lapatinib, in HER-2 overexpressing breast cancer cells. Combination proliferation assays were performed in two HER-2 positive breast cancer cell lines, SKBR-3 and BT-474. Trastuzumab combined with lapatinib was also tested in BT-474 xenografts. In proliferation assays, dual targeting with trastuzumab and gefitinib or lapatinib showed synergy or additivity in both SKBR-3 and BT-474 cells. Trastuzumab (10 nM) or gefitinib (5 μM) alone did not induce significant apoptosis, whereas lapatinib (0.75 μM) induced significant apoptosis in SKBR-3 cells. Trastuzumab combined with lapatinib further enhanced apoptosis induction. Trastuzumab (10 nM) and gefitinib (5 μM) induced apoptosis comparable to lapatinib alone (0.75 μM), suggesting that inhibition of both EGFR and HER-2 may be required to induce apoptosis in these cells. Pre-treatment with trastuzumab and gefitinib or lapatinib enhanced response to chemotherapy in vitro. The combination of trastuzumab and lapatinib also effectively blocked tumour growth in vivo. Dual targeting of EGFR and HER-2, by combining trastuzumab with EGFR/HER-2 tyrosine kinase inhibitors, may improve response in HER-2 overexpressing breast cancer cells that also express EGFR.     

Bee venom induces apoptosis through caspase-3 activation in synovial fibroblasts of patients with rheumatoid arthritis.

Bee venom (BV) has been used traditionally for the control of pain and inflammation in various chronic inflammatory diseases, including rheumatoid arthritis (RA) in Oriental medicine. However, it is still unclear how BV exerts its beneficial effects on the clinical course of RA patients. To investigate the effect of BV on the treatment of rheumatoid synovitis, we examined the inhibition of cell growth and induction of apoptosis in human rheumatoid synovial fibroblasts. Rheumatoid synovial fibroblasts were surgically obtained from patients with RA. Cell proliferation and viability were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis of synovial cells treated with 10 microg/ml BV for 24 h was identified by 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, DNA fragmentation assay, RT-PCR, and Western blot analysis. It was demonstrated that rheumatoid synovial cells treated with 10 microg/ml BV for 24 h exhibited apoptotic features and fragmentation of DNA. In addition, BV induces apoptosis in rheumatoid synovial cells through a decrease in BCL2 expression and an increase in BAX and caspase-3 (CASP3) expression. It is suggested that BV inhibits the proliferation of rheumatoid synovial cells through induction of apoptosis by CASP3 activation.     

Dissimilar effects of curcumin on human granulosa cells: Beyond its anti-oxidative role

Global infertility prevalence has been increasing in recent decades, mainly due to advanced reproductive age. Concerned women look for dietary supplements with antioxidant properties advertised as a natural way to increase fertility. Curcumin (CUR) is a polyphenol with antioxidant and anti-inflammatory properties. CUR elicits apoptotic cell death as evidenced in some tumor cells. In this work, the effect of CUR on granulosa cells (GC) was studied. GC surround the oocyte, providing nutrient exchange and hormone production, necessary for its development. COV434 cell line and primary human granulosa cells (hGC) cultures from patients undergoing Assisted Reproductive Technology (ART) were used. GC were treated with CUR (0.001−50 μM) at different times (24−72 h). Low concentrations of CUR showed an increase on cell viability. Likewise, it leads to a decrease in ROS/RNS formation after stress induction, suggesting a protective role. Changes in hormonal levels were not observed. In contrast, high concentrations of CUR triggered a reduction on cell viability and a programmed cell death mechanism. Allied to the above results, high doses of CUR affected hormonal function of GCs. Our work reinforces the benefits of dietary supplements, namely CUR, on the main functions of GC and, consequently, on reproductive success.     

Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes.     

DI-(2-ethylhexyl) phthalate-induced cell proliferation is involved in the inhibition of gap junctional intercellular communication and blockage of apoptosis in mouse Sertoli cells

Di-(2-ethylhexyl) phthalate (DEHP) has been studied on gap junctional intercellular communication (GJIC) and apoptosis in cultured normal mouse Sertoli cells. Since the inhibition of GJIC and programmed cell death or apoptosis play important roles in tumor promotion and developmental toxicity, it has been hypothesized that tumor promoters may inhibit apoptosis by blocking GJIC. The results showed that the most significant downregulation of GJIC was detected at 9 h after DEHP treatment. However, a significant concentration-dependent pattern was not observed at concentrations of 100 and 500 microM, but there was a time-dependent recovery of GJIC. DEHP inhibited the apoptotic changes in the cells such as chromatin condensation, nuclear fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Morphological changes related to apoptosis appeared in the nontreated cells after 12 h of serum deprivation. These morphological changes were significantly reduced in the TM5 Sertoli cells treated with 500 microM DEHP for 24 h. These results suggest that DEHP inhibited apoptosis in this cell line, preceded by the downregulation of GJIC. It was also found that DEHP reduced the phosphorylation of Cx43, which might partly explain the mechanism of inhibition of GJIC.     

The role of inhibition of gap junctional intercellular communication in rodent liver tumor induction by phthalates: review of data on selected phthalates and the potential relevance to man

Inhibition of gap junctional intercellular communication (GJIC) has been postulated as a nongenotoxic carcinogenic mechanism, probably related to tumor promotion. Recent studies assessed the role of GJIC in the induction of rodent liver tumors by high levels of phthalate esters. Studies with di(2-ethylhexyl) (DEHP) and diisononyl (DINP) phthalates demonstrated that inhibition of GJIC in rats and mice was well correlated with induction of both liver tumors and markers for peroxisomal proliferation. However, GJIC was unaffected in hamsters and primates, species in which phthalate treatment does not induce peroxisomal proliferation. In vitro studies which extended the database to include human liver cells mirrored the in vivo situation; GJIC was inhibited in rat and mouse cells but not in cells from unresponsive species including humans. Peroxisomal proliferation has been characterized as a species-specific process essential for phthalate-induced rodent liver tumor induction. That GJIC was not inhibited in primate liver or human liver cells provides evidence for a second species-specific carcinogenic process. Thus the GJIC data along with those from studies of peroxisomal proliferation support the view that the carcinogenic effects of DEHP and DINP in rodents are not relevant to humans.     

Resveratrol inhibits oral squamous cell carcinoma cells proliferation while promoting apoptosis through inhibition of CBX7 protein

As a natural compound, resveratrol (Res) is confirmed to be promising drug for the treatment of malignant tumors. Therefore, our study aimed to observe the impacts of Res on the proliferation and apoptosis of oral squamous cell carcinoma cells (HSC‐3 cells) as well as the mechanism involving chromobox protein homolog 7 (CBX7) signal transduction. HSC‐3 cells were treated with Res, Akt agonist (AL3818) and p16 inhibitor (SC79), and transfected with CBX7 mimics and inhibitor plasmids. The CCK‐8 assay was used to detect cell proliferation, flow cytometry was performed to assess cell cycle and apoptosis, and cell colonies and histone DNA level were also measured. Western blot analysis was used to determine the expression levels of related proteins. HSC‐3 cells showed decreased cell proliferation, colonies, BrdU‐counled cells and increased apoptosis, histone DNA level, the activities of caspase‐3 and caspase‐9 when treated with Res. Western blot analysis revealed elevated Cle‐PARP and Cle‐caspase 3 expression and reduced t‐PARP expression in HSC‐3 cells treated with Res compared with control. AL3818 and SC79 could decrease the inhibitory effects of Res on the growth of HSC‐3 cells. Furthermore, CBX7 overexpression could also partly reverse the roles of Res in the growth of HSC‐3 cells, and Akt and p16 signal transduction. Our results demonstrate that Res suppresses the proliferation, and induces the apoptosis of oral squamous cell carcinoma cells through the inhibition of CBX7/Akt and the activation of p16 cascades.     

The role of transcription factor Nrf2 in the toxicity of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in C57BL/6 mouse astrocytes

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are members of perfluoroalkyl substances (PFAS). This study aimed to determine the protective role of Nrf2 against the toxicity of these agents. Nrf2-/- and wild-type astrocytes were exposed to PFOS (75−600 μM) and PFOA (400−1000 μM) for 24 h. Lactate dehydrogenase (LDH) release was significantly higher in nrf2-/- than in the wild-type astrocytes. Exposure to 600 μM PFOS and 800 μM PFOA showed significant increases in reactive oxygen species, lipid peroxidation, and apoptosis in nrf2-/- astrocytes as compared to wild-type astrocytes. The GSH/GSSG ratio was significantly decreased in nrf2-/- astrocytes when compared to wild-type astrocytes. Additionally, PFOS and PFOS caused dramatic ultrastructural alterations to the mitochondria. BHT pretreatment in wild-type cells decreased ROS production with exposure to both agents. Results of the present study conclude that PFOS and PFOA are cytotoxic to astrocytes and that nrf2 -/- cells are more sensitive to toxicity by these agents.     

Perfluorinated compounds differentially affect steroidogenesis and viability in the human adrenocortical carcinoma (H295R) in vitro cell assay

Perfluorinated compounds (PFCs) comprise a large class of man-made chemicals of which some are persistent and present throughout the ecosystem. This raises concerns about potential harmful effects of such PFCs on humans and the environment. In order to investigate the effects of potentially harmful PFCs on steroid hormone production, human adrenocortical H295R cells were exposed to three persistent PFCs including perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) at six different concentrations (6 nM to 600 μM) for 48 h. Exposure to 600 μM PFOS resulted in a dose-responsive increase in oestradiol as well as a smaller dose-responsive increase in progesterone and testosterone secretion measured using radioimmunoassay. The aromatase activity was not significantly altered by PFOS. Only small changes in hormone secretion were detected following exposure to PFOA and PFNA. Gene expression of CYP11A, quantified using qRT-PCR was decreased by all exposure doses of PFOA, whereas HMGR expression was decreased by 60 nM PFNA. The viability markedly decreased by exposure to 600 μM of PFOA or PFNA, but not PFOS. Flow cytometric analysis demonstrated a significant increase in apoptosis following exposure to PFNA at the highest concentration. We conclude that PFOS is capable of altering steroidogenesis in the H295R in vitro model by a mechanism other than changes in gene expression or activity of aromatase. Additionally, PFCs appear to differentially affect cell viability with induction of cell death via apoptosis at high doses of PFNA.     

The benzene metabolite trans,trans-muconaldehyde blocks gap junction intercellular communication by cross-linking connexin43

Benzene is used at large volumes in many different human activities. Hematotoxicity and cancer-causation as a result of benzene exposure was recognized many years ago, but the mechanisms involved remain unclear. Aberrant regulation of gap junction intercellular communication (GJIC) has been linked to both cancer induction and interference with normal hematopoietic development. We have previously suggested that inhibition of GJIC may play a role in benzene toxicity since benzene metabolites were found to block GJIC, the ring-opened trans,trans-muconaldehyde (MUC) being the most potent metabolite. In the present work we have studied the molecular mechanisms underlying the MUC-induced inhibition of gap junctional communication. We show that MUC induces cross-linking of the gap junction protein connexin43 and that this is likely to be responsible for the induced inhibition of GJIC, as well as the loss of connexin43 observed in Western blots. We also show that glutaraldehyde possesses similar effects as MUC, and we compare the effects to that of formaldehyde. The fact that glutaraldehyde and formaldehyde have been associated with induction of leukemia as well as disturbance of hematopoiesis, strengthens the possible link between the effect of MUC on gap junctions, and the toxic effects of benzene.     

Differences between serum polar lipid profiles of male and female rheumatoid arthritis patients in response to glucocorticoid treatment

Objective

As there are pharmacological differences between males and females, and glucocorticoid (GC) treatment is associated with increased cardiovascular mortality rate in rheumatoid arthritis (RA) patients, it is important to study serum polar lipid profiles of male and female patients in response to GC therapy. Gender differences may require an adjustment to the treatment strategy for a selection of patients.

Methods

Serum samples from 281 RA patients were analysed using a targeted lipidomics platform. The differences in GC use and gender on polar lipid profiles were cross sectionally examined by multiple linear regressions, while correcting for confounding factors.

Results

Differences in polar lipids between GC users and non-GC users in females and males were merely restricted to lysophospholipids (lysophosphatidylcholines and lysophosphatidylethanolamines). Lysophospholipids in female patients treated with GCs were significantly higher than female patients not treated with GCs (p = 6.0 E?6), whereas no significant difference was observed in male GC users versus non-users (p = 0.397).

Conclusion

The lysophospholipid profiles in response to GCs were significantly different between male and female RA patients, which may have implications for the cardiovascular risk of GC treatment.

     

Inhibition of gap-junctional-intercellular communication in intact rat liver by nongenotoxic hepatocarcinogens

Many nongenotoxic hepatocarcinogens can induce cell proliferation, and inhibit apoptosis and gap-junctional-intercellular communication (GJIC). GJIC, the movement of small molecules (less than 1.2 kD) through membrane channels, is important in regulating cellular homeostasis and differentiation. The inhibition of hepatic GJIC can increase cell proliferation and possibly, inhibit apoptosis. In this study, the relationship between hepatic GJIC, proliferation, and apoptosis was examined in rats treated for 7 days with tumor-promoting doses of the nongenotoxic hepatocarcinogens phenobarbital (PB; 800 ppm), pregnenolone-16alpha-carbonitrile (PCN; 1000 ppm), and Aroclor 1254 (PCB; 100 ppm). In addition, 3-methylcholanthrene (3MC) was included as a negative control. PB, PCN, and PCB increased parenchymal-cell proliferation and inhibited hepatic apoptosis, while no alteration in these growth parameters was observed in 3MC-treated rats. GJIC, as measured by fluorescent-dye transfer through intact liver, was decreased nearly 50% by PB, PCN, and PCB, yet no effect on GJIC was observed in liver from 3MC-treated rats. These data indicate that compounds that inhibit GJIC in liver may be nongenotoxic hepatocarcinogens, which occurs simultaneously during increased cell proliferation and inhibited apoptosis.     

Dexamethasone differentially regulates Bcl-2 family proteins in human proliferative chondrocytes: Role of pro-apoptotic Bid

Glucocorticoids (GCs) are widely used to treat inflammatory diseases and cancers. A multitude of undesired side effects have been reported in GC-treated patients including decreased linear bone growth. We have previously reported that GCs activate the caspase cascade and trigger Bax-mediated mitochondrial apoptosis in growth plate chondrocytes causing growth retardation in young mice. To further explore the role of mitochondrial apoptosis in GC-induced bone growth retardation, a number of pro- and anti-apoptotic proteins were studied in ex vivo cultures of human growth plate cartilage and human HCS-2/8 proliferative chondrocytes exposed to dexamethasone. Dexamethasone was found to increase the pro-apoptotic proteins Bcl-xS, Bad, and Bak as well as the proteolysis of Bid. Anti-Bid small interfering RNA partially rescued the chondrocytes from dexamethasone-induced apoptosis. Taken together, our data suggest that GC treatment differentially regulates Bcl-2 family member proteins to facilitate mitochondrial apoptosis in proliferative chondrocytes thereby contributing to GC-induced bone growth impairment. Prevention of this imbalance between pro- and anti-apoptotic Bcl-2 family proteins may provide a new strategy to protect from adverse effects of GCs on bone growth.