Sodium arsenite-induced cytotoxicity is regulated by BNIP3L/Nix-mediated endoplasmic reticulum stress responses and CCPG1-mediated endoplasmic reticulum-phagy.

We elucidated the BNIP3L/Nix and SQSTM1/p62 molecular mechanisms in sodium arsenite (NaAR)-induced cytotoxicity. Considerable changes in the morphology and adhesion of H460 cells were observed in response to varying NaAR concentrations. NaAR exposure induced DNA damage-mediated apoptosis and Nix accumulation via proteasome inhibition. Nix targets the endoplasmic reticulum (ER), inducing ER stress responses. p62 and Nix were colocalized and their expressions were inversely correlated. Autophagy inhibition upregulated Nix, p62, cell cycle progression gene 1 (CCPG1), heme oxygenase (HO)- 1, and calnexin expression. Nix knockdown decreased the NaAR-induced ER stress and microtubule-associated protein 1 A/1B light-chain 3 (LC3) B-II levels and increased the CCPG1 and calnexin levels. p62 knockdown upregulated Nix, LC3-II, and CCPG1 expressions and the ER stress responses, indicating that p62 regulates Nix levels. Nix downstream pathways were mitigated by Ca2+ chelators. We demonstrate the critical roles of Nix and p62 in ER stress and ER-phagy in response to NaAR.

Arsenite-induced cytotoxicity is regulated by poly-ADP ribose polymerase 1 activation and parthanatos in p53-deficient H1299 cells: The roles of autophagy and p53.

Arsenic is a double-edged sword metalloid since it is both an environmental carcinogen and a chemopreventive agent. Arsenic cytotoxicity can be dependent or independent of the tumor suppressor p53. However, the effects and the underlying molecular mechanisms of arsenic cytotoxicity in p53-deficient cells are still unclear. Here, we report a distinctive cell death mode via PARP-1 activation by arsenic in p53-deficient H1299 cells. H1299 (p53-/-) cells showed higher sensitivity to sodium arsenite (NaAR) than H460 (p53+/+) cells. H460 cells induced canonical apoptosis through caspase-dependent poly-ADP ribose polymerase 1 (PARP-1) cleavage and induced the expression of phospho-p53 and p21. However, H1299 cells induced poly-ADP-ribose (PAR) polymer accumulation and caspase-independent parthanatos, which was inhibited by 3-aminobenzamide (AB) and nicotinamide (NAM). Fractionation studies revealed the mitochondrial translocation of PAR polymers and nuclear translocation of the apoptosis-inducing factor (AIF). Although the exposure of NaAR to p53-overexpressing H1299 cells increased the PAR polymer levels, it inhibited parthanatos by inducing p21 and phospho-p53 expression. LC3-II and p62 accumulated in a NaAR dose- and exposure time-dependent manner, and this accumulation was further enhanced by autophagy inhibition, indicating that arsenic inhibits autophagic flux. p53 overexpression led to a decrease in the p62 levels, an increase in the LC3-II levels, and reduced parthanatos, indicating that arsenic induces p53-dependent functional autophagy. These results show that the NaAR-induced cytotoxicity in p53-deficient H1299 cells is regulated by PARP-1 activation-mediated parthanatos, which is promoted by autophagy inhibition. This suggests that PARP-1 activation could be used as an effective therapeutic approach for arsenic toxicity in p53-deficient cells.

The role of autophagy in cadmium-induced acute toxicity in glomerular mesangial cells and tracking polyubiquitination of cytoplasmic p53 as a biomarker.

Cadmium (Cd) is a highly toxic environmental pollutant that can severely damage the kidneys. Here, we show that Cd-induced apoptosis is promoted by the cytoplasmic polyubiquitination of p53 (polyUb-p53), which is regulated by the polyubiquitination of SQSTM1/p62 (polyUb-p62) and autophagy in mouse kidney mesangial cells (MES13E cells). p53 was detected in monomeric and different high-molecular-weight (HMW) forms after Cd exposure. Monomeric p53 levels decreased in a concentration- and time-dependent manner. HMW-p53 transiently accumulated in the cytoplasm independent of proteasome inhibition. The expression patterns of p53 were similar to those of p62 upon Cd exposure, and the interactions between polyUb-p53 and polyUb-p62 were observed using immunoprecipitation. P62 knockdown reduced polyUb-p53 and upregulated nuclear monomeric p53, whereas p53 knockdown reduced polyUb-p62. Autophagy inhibition induced by ATG5 knockdown reduced Cd-induced polyUb-p62 and polyUb-p53 but upregulated the levels of nuclear p53. Pharmacological inhibition of autophagy by bafilomycin A1 increased polyUb-p62 and polyUb-p53 in the cytoplasm, indicating that p53 protein levels and subcellular localization were regulated by polyUb-p62 and autophagy. Immunoprecipitation and immunofluorescence revealed an interaction between p53 and LC3B, indicating that p53 was taken up by autophagosomes. Cd-resistant RMES13E cells and kidney tissues from mice continuously injected with Cd had reduced polyUb-p53, polyUb-p62, and autophagy levels. Similar results were observed in renal cell carcinoma cell lines. These results indicate that cytoplasmic polyUb-p53 is a potential biomarker for Cd-induced acute toxicity in mesangial cells. In addition, upregulation of nuclear p53 may protect cells against Cd cytotoxicity, but abnormal p53 accumulation may contribute to tumor development.

Arsenite-induced cytotoxicity is regulated by p38-SQSTM1/p62 and JNK-BNIP3L/Nix signaling in lung cancer cells.

Arsenic is a potent carcinogen in humans. However, the molecular mechanisms underlying its toxicity in lung cancer remain unclear. Here, we report that arsenite-induced cytotoxicity is regulated by SQSTM1/p62 and BNIP3L/Nix signaling in non-small-cell lung cancer H460 cells. Arsenite exposure resulted in dose-dependent growth inhibition, which was associated with apoptosis, as demonstrated by depolarized mitochondrial membrane potential and cleavage of caspase-8, caspase-3, PARP-1, and Bax. The autophagy adaptor p62 was detected in the monomeric and multiple high-molecular-weight (HMW) forms, and protein levels were upregulated depending on both arsenite concentrations (≤45 µM) and exposure times (<24 h). LC3-II, an autophagy marker, was upregulated as early as 1 h after arsenite treatment. Expression of Nix, a mitochondrial outer membrane protein, continued to increase with arsenite concentration and exposure time; it was detected in the monomeric and multiple HMW forms. Soon after arsenite exposure, p62 colocalized with Nix in the cytoplasm, and p62 knockdown reduced the Nix levels and increased the LC3-II levels. In contrast, Nix knockdown did not affect the p62 and LC3-II levels but reduced caspase-8, caspase-3, and Bax cleavage, indicating that Nix accumulation resulted from its reduced autophagic degradation and promoted apoptosis. p38 inhibition markedly increased arsenite-induced Nix protein and reduced p62 protein levels, resulting in increased autophagy and apoptosis. Furthermore, c-Jun NH2-terminal kinase inhibition reduced Nix and Bax cleavage, and both signaling pathways were suppressed by N-acetylcysteine treatment. Our results suggest that arsenite-induced cytotoxicity is modulated by the coordinated action of p62 and Nix through MAPK.

Sec-O-glucosylhamaudol mitigates inflammatory processes and autophagy via p38/JNK MAPK signaling in a rat neuropathic pain model.

BACKGROUND: This study investigated the effect of intrathecal Sec-O-glucosylhamaudol (SOG) on the p38/c-Jun N-terminal kinase (JNK) signaling pathways, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related inflammatory responses, and autophagy in a spinal nerve ligation (SNL)-induced neuropathic pain model. METHODS: The continuous administration of intrathecal SOG via an osmotic pump was performed on male Sprague-Dawley rats (n = 50) with SNL-induced neuropathic pain. Rats were randomized into four groups after the 7th day following SNL and treated for 2 weeks as follows (each n = 10): Group S, sham-operated; Group D, 70% dimethylsulfoxide; Group SOG96, SOG at 96 µg/day; and Group SOG192, SOG at 192 µg/day. The paw withdrawal threshold (PWT) test was performed to assess neuropathic pain. Western blotting of the spinal cord (L5) was performed to measure changes in the expression of signaling pathway components, cytokines, and autophagy. Additional studies with naloxone challenge (n = 10) and cells were carried out to evaluate the potential mechanisms underlying the effects of SOG. RESULTS: Continuous intrathecal SOG administration increased the PWT with p38/JNK mitogen-activated protein kinase (MAPK) pathway and NF-κB signaling pathway inhibition, which induced a reduction in proinflammatory cytokines with the concomitant downregulation of autophagy. CONCLUSIONS: SOG alleviates mechanical allodynia, and its mechanism is thought to be related to the regulation of p38/JNK MAPK and NF-κB signaling pathways, associated with autophagy during neuroinflammatory processes after SNL.

Salinomycin suppresses TGF-ß1-induced EMT by down-regulating MMP-2 and MMP-9 via the AMPK/SIRT1 pathway in non-small cell lung cancer.

Salinomycin (Sal) is a recently identified anti-tumor drug for treating several types of solid tumor; however, its effects on the migratory and invasive properties of non-small cell lung cancer (NSCLC) remain unclear. This study investigated the inhibitory effect underlying mechanisms of Salon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-to-mesenchymal transition (EMT) and cell migration. Sal solidly blocked cell migration and invasion enhancement by TGF-ß1-induced EMT, through recovering E-cadherin loss and suppressing mesenchymal markers induction, as well as TGF-ß1-mediated AMPK/SIRT signaling activity upregulation. The pharmacologic inhibition or knockdown of AMPK or SIRT1 can act synergistically with Sal to inhibit TGF-ß1-induced MMP-2 and MMP-9. In contrast, AMPK or SIRT1 upregulation can protect against TGF-ß1-induced MMP-2 and MMP-9 inhibition by Sal. Next we demonstrated that the MMP-2 and MMP-9 knockdown can act synergistically with Sal to inhibit TGF-ß1-induced EMT. Moreover, treatment of PMA of MMP activator increased TGF-ß1-induced MMP-2 and MMP-9, even with Sal. Our results demonstrate that Sal suppresses TGF-ß1-induced EMT by downregulating MMP-2 and MMP-9 through the AMPK/SIRT pathway, thereby inhibiting lung cancer cell migration and invasion.

Cytoplasmic sirtuin 6 translocation mediated by p62 polyubiquitination plays a critical role in cadmium-induced kidney toxicity.

Sirtuin 6 (Sirt6) is important for maintaining kidney homeostasis and function. Cd exposure increases the risk of developing kidney diseases. However, the role of Sirt6 in kidney disease mechanisms is unclear. Here, we evaluated the role of Sirt6 in Cd-induced kidney toxicity. After Cd exposure, p62/sequestosome-1 (SQSTM1), an autophagy substrate, accumulated in mouse kidney mesangial cells in monomeric and polyubiquitinated (polyUb) forms. Sirt6 accumulated in response to Cd treatment at concentrations below the half-maximal inhibitory concentration and decreased after 12 h of treatment. Sirt6 and p62 co-localized in the nucleus and redistributed to the cytosol after Cd treatment. Sirt6 was mainly present in nuclei-rich membrane fractions. Sirt6 interacted with p62. Ub, and microtubule-associated protein light chain 3 (LC3). Knockdown of p62 promoted Sirt6 nuclear accumulation and inhibited apoptosis. Sirt6 overexpression altered levels of polyUb-p62 and apoptosis. At earlier times during Cd treatment, polyubiquitination of p62 and apoptosis were reduced. Cytoplasmic translocation of Sirt6 occurred later, with increased polyubiquitination of p62 and apoptosis. Bafilomycin 1 (BaF1) treatment promoted cytosolic Sirt6 accumulation, increasing cell death. Silencing autophagy related 5 (Atg5) increased nuclear Sirt6 levels, reduced polyUb-p62, and inhibited cell death, indicating that autophagy was necessary for Sirt6 redistribution. Cd resistance was associated with reduced polyUb-p62 and persistent Sirt6 expression. Cd treatment in mice for 4 weeks promoted p62, Sirt6, and LC3-II accumulation, inducing apoptosis in kidney tissues. Overall, our findings show that polyUb-p62 targeted Sirt6 to autophagosomes, playing a crucial role in Cd-induced cell death and kidney damage.

Autophagy-mediated cytoplasmic accumulation of p53 leads to apoptosis through DRAM-BAX in cadmium-exposed human proximal tubular cells.

The tumor suppressor p53 is involved in cadmium (Cd)-induced apoptosis and autophagy. However, the regulatory mechanisms of p53 in Cd-induced kidney injury are not well established. Here, we report the role of autophagy in Cd-induced p53 induction in human proximal tubular cells (HK-2). HK-2 cells treated with Cd induced the expression of p53, DNA damage autophagy modulator (DRAM), and Bcl-2-associated X protein (BAX), as well as caused poly [ADP-ribose] polymerase 1 (PARP-1) cleavage. Cd exposure also induced autophagy with the accumulation of monomeric p62 and multiple high molecular weight form (HMW)-p62. The expression levels of p53, p62, microtubule-associated protein 1A/1B-light chain 3 (LC3)-1, and LC3-II were similar in the sense that they increased up to 12 h and then gradually decreased. DRAM and BAX levels began to increase post autophagy induction and continued to increase, indicating that autophagy preceded apoptosis. While the genetic knockdown of p53 downregulated HWM-p62, DRAM, and BAX, the expression levels of these proteins were upregulated by p53 overexpression. The genetic knockdown of p62 downregulated p53, autophagy, DRAM, and BAX. The inhibition of autophagy through pharmacological and genetic knockdown reduced p53 and inhibited Cd-induced apoptosis. Collectively, Cd induces apoptosis through p53-mediated DRAM-BAX signaling, which can be regulated by autophagy.

Correction to: Cytoplasmic sirtuin 6 translocation mediated by p62 polyubiquitination plays a critical role in cadmium-induced kidney toxicity.

In the original publication the grant number is incorrectly published.

Gabexate mesilate ameliorates the neuropathic pain in a rat model by inhibition of proinflammatory cytokines and nitric oxide pathway via suppression of nuclear factor-κB.

BACKGROUND: This study examined the effects of gabexate mesilate on spinal nerve ligation (SNL)-induced neuropathic pain. To confirm the involvement of gabexate mesilate on neuroinflammation, we focused on the activation of nuclear factor-κB (NF-κB) and consequent the expression of proinflammatory cytokines and inducible nitric oxide synthase (iNOS). METHODS: Male Sprague-Dawley rats were used for the study. After randomization into three groups: the sham-operation group, vehicle-treated group (administered normal saline as a control), and the gabexate group (administered gabexate mesilate 20 mg/kg), SNL was performed. At the 3rd day, mechanical allodynia was confirmed using von Frey filaments, and drugs were administered intraperitoneally daily according to the group. The paw withdrawal threshold (PWT) was examined on the 3rd, 7th, and 14th day. The expressions of p65 subunit of NF-κB, interleukin (IL)-1, IL-6, tumor necrosis factor-α, and iNOS were evaluated on the 7th and 14th day following SNL. RESULTS: The PWT was significantly higher in the gabexate group compared with the vehicle-treated group (P < 0.05). The expressions of p65, proinflammatory cytokines, and iNOS significantly decreased in the gabexate group compared with the vehicle-treated group (P < 0.05) on the 7th day. On the 14th day, the expressions of p65 and iNOS showed lower levels, but those of the proinflammatory cytokines showed no significant differences. CONCLUSIONS: Gabexate mesilate increased PWT after SNL and attenuate the progress of mechanical allodynia. These results seem to be involved with the anti-inflammatory effect of gabexate mesilate via inhibition of NF-κB, proinflammatory cytokines, and nitric oxide.

Poly-ubiquitinated p62/SQSTM1-mediated hemeoxygenase-1 stabilization plays a critical role in cadmium-induced apoptosis of mouse monocyte Raw264.7 cells.

Cadmium (Cd) is a toxic heavy metal that can affect many organs, leading to serious pathological disorders through immune suppression. Here, we investigated the molecular mechanisms underlying the response of monocytes to Cd exposure. Cd treatment of Raw264.7 cells activated antioxidant enzymes, such as hemeoxygenase-1 (HO-1), superoxide dismutase, and catalase. Cd exposure upregulated p53, p53 phosphorylation, p21, and γH2AX phosphorylation. Cd exposure also induced poly ADP-ribose polymerase 1 (PARP-1) cleavage. These findings indicated that Cd induces apoptosis through oxidative stress-mediated DNA damage. Furthermore, upregulation of microtubule-associated protein 1 light chain 3B-II (LC3B-II), an indicator of autophagy, was found to depend on Cd concentration. Accumulation of an autophagy substrate p62/SQSTM1 in monomeric p62 and polyubiquitinated (polyUb)-p62 forms, was suppressed upon N-acetylcysteine treatment Cd-exposed Raw264.7 cells, indicating an impairment of autophagic degradation during oxidative stress. Knockdown of p62 in Raw264.7 cells using small interfering RNA (siRNA) downregulated HO-1 expression and reduced apoptosis. HO-1 knockdown suppressed apoptosis by decreasing the poly-ubiquitination of p62. Treatment with hemin and MG132 enhanced Cd-mediated increases in HO-1 and polyUb-p62 levels, resulting in increased apoptosis, which indicated that Cd-induced HO-1 accumulation is associated with polyUb-p62 formation. p62 and HO-1 interactions were demonstrated by immunofluorescence and immunoprecipitation assays. Additionally, p62 was downregulated in Raw264.7 cells in response to H2O2 and a low level of HO-1 was induced. Cells that were highly sensitive to Cd did not form polyUb-p62, resulting in insufficient HO-1 accumulation. These results suggest that maintenance of HO-1 stability via poly-ubiquitination of p62 in Cd-exposed monocytes promotes apoptosis, which could be involved in immune suppression.

Polyubiquitination of p62/SQSTM1 is a prerequisite for Fas/CD95 aggregation to promote caspase-dependent apoptosis in cadmium-exposed mouse monocyte RAW264.7 cells.

Cadmium(Cd) induces cytotoxicity via autophagy-induced apoptosis in non-activated mouse monocytes; however, the molecular mechanism remains unclear. Here, we show that autophagy induces Fas (CD95/APO-1)-mediated apoptosis by promoting accumulation of p62/SQSTM1 in response to Cd. Cd produced tumor necrosis factor (TNF)-α, peaking at 6 h, and exhibiting a concentration-dependent increase. Immunoblot analysis revealed polyubiquitinated (polyUb) full-length Fas (antibody clone G-9) and reduced cytosolic Fas (antibody clone M-20) in Cd-exposed RAW264.7 cells. The accumulation of polyUb-Fas was transient and positively correlated with polyUb-p62 and polyUb-proteins. Autophagy inhibition via chemical and genetic modulation suppressed Cd-induced polyUb-p62, polyUb-Fas, and polyUb-protein levels, whereas the level of cytosolic Fas recovered to that of the control. Immunofluorescence (IF) staining for full-length Fas, p62, and ubiquitin revealed an aggregated pattern in Cd-induced apoptotic cells, which was inhibited by blocking autophagy. Fas colocalized with microtubule-associated protein 1 light chain (LC)-3B. IF staining and immunoprecipitation assays revealed colocalization and interaction among p62, Ub, and Fas. Knockdown of p62 reduced the binding of Ub and Fas. Together, these data suggest that polyUb-p62 targets Fas and recruits it to autophagosomes, where Fas transiently aggregates to promote apoptosis and is degraded with polyUb-p62. In conclusion, autophagy regulates C-terminal cytosolic Fas aggregation via p62 polyubiquitination, which is required for apoptosis and may play a critical role in the production of select cytokines.

Effect of sec-O-glucosylhamaudol on mechanical allodynia in a rat model of postoperative pain.

BACKGROUND: This study was performed in order to examine the effect of intrathecal sec-O-glucosylhamaudol (SOG), an extract from the root of the Peucedanum japonicum Thunb., on incisional pain in a rat model. METHODS: The intrathecal catheter was inserted in male Sprague-Dawley rats (n = 55). The postoperative pain model was made and paw withdrawal thresholds (PWTs) were evaluated. Rats were randomly treated with a vehicle (70% dimethyl sulfoxide) and SOG (10 µg, 30 µg, 100 µg, and 300 µg) intrathecally, and PWT was observed for four hours. Dose-responsiveness and ED50 values were calculated. Naloxone was administered 10 min prior to treatment of SOG 300 µg in order to assess the involvement of SOG with an opioid receptor. The protein levels of the δ-opioid receptor, κ-opioid receptor, and µ-opioid receptor (MOR) were analyzed by Western blotting of the spinal cord. RESULTS: Intrathecal SOG significantly increased PWT in a dose-dependent manner. Maximum effects were achieved at a dose of 300 µg at 60 min after SOG administration, and the maximal possible effect was 85.35% at that time. The medial effective dose of intrathecal SOG was 191.3 µg (95% confidence interval, 102.3-357.8). The antinociceptive effects of SOG (300 µg) were significantly reverted until 60 min by naloxone. The protein levels of MOR were decreased by administration of SOG. CONCLUSIONS: Intrathecal SOG showed a significant antinociceptive effect on the postoperative pain model and reverted by naloxone. The expression of MOR were changed by SOG. The effects of SOG seem to involve the MOR.

Nefopam downregulates autophagy and c-Jun N-terminal kinase activity in the regulation of neuropathic pain development following spinal nerve ligation.

BACKGROUND: Neurodegeneration is associated with changes in basal cellular function due to the dysregulation of autophagy. A recent study introduced the involvement of autophagy during spinal nerve ligation (SNL). Nefopam has shown potential for reducing neuropathic pain, but the underlying mechanisms are unknown. Here, we investigated the effects of nefopam on neuropathic pain development following SNL, focusing on the involvement of autophagy. METHODS: The functional role of nefopam in capsaicin-induced autophagy was assessed by human glioblastoma M059 K cells. The neuropathic pain model was used to determine whether the effect of nefopam on pain control was mediated through autophagy control. Neuropathic pain was induced by L5 and L6 SNL in male rats randomized into three groups: Group S (sham-operated), Group C (received normal saline), and Group E (received nefopam). A behavioral test using a von Frey was examined. Expression changes of autophagy in response to nefopam was analyzed in spinal cord tissues (L4-L6) by immunoblotting and immunohistochemistry. RESULTS: The paw withdrawal threshold examined on days 3, 5, 7, and 14 post-SNL was significantly higher in Group E than in Group C. SNL increased the levels of microtubule-associated protein 1 light chain 3B (LC3B-1), with concomitant reduction of sequestosome 1 (SQTSM1/p62), compared with Group S, indicating that SNL induced autophagy. These effects were reversed by nefopam injection, and the results were confirmed by immunohistochemistry for LC3-I/II. Furthermore, SNL-mediated JNK activation was markedly decreased following nefopam injection. Hematoxylin and eosin staining on Day 14 post-SNL revealed that SNL caused lymphocyte infiltration and oligodendrocyte localization in the substantia gelatinosa of the dorsal gray horn, which were reduced by nefopam injection. CONCLUSION: Collectively, the mode of action of nefopam on neuropathic pain appears to be associated with downregulation of phospho-JNK and autophagy, as well as modulation of the immune response.

The critical role of autophagy in cadmium-induced immunosuppression regulated by endoplasmic reticulum stress-mediated calpain activation in RAW264.7 mouse monocytes.

Cadmium (Cd) has toxic and suppressive effects on the immune system, but the underlying mechanisms remain poorly understood. Here, we show that autophagy plays a critical role in regulation of Cd-induced immunosuppression in RAW264.7 cells. Cd decreased cell viability in a dose-dependent manner; cleaved caspase-8, caspase-3, and poly (ADP-ribose) polymerase (PARP)-1; increased DNA laddering; induced CCAAT-enhancer-binding protein homologous protein (CHOP); and reduced tumor necrosis factor (TNF)-α expression; indicating that caspase-dependent and endoplasmic reticulum (ER)-mediated apoptosis are involved in Cd-induced immunotoxicity. Furthermore, Cd induced autophagy, as demonstrated by microtubule-associated protein 1 light chain 3B (LC3B) plasmid DNA transfection and its conversion from LC3-I to the LC3-II form by autophagy inhibitors, via AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling. Pharmacological and genetic inhibition of autophagy suppressed Cd-induced apoptosis, as evidenced by inhibition of caspase-8, caspase-3, and PARP-1 cleavage, indicating that autophagy promotes apoptosis. The pan-caspase inhibitor zVAD inhibited Cd-induced apoptosis, but increased autophagy and decreased cell viability, indicating that autophagy can compensate for reduced apoptotic cell death. Calpain inhibitors blocked Cd-induced apoptosis and autophagy, indicating that calpain plays a critical role in Cd cytotoxicity. Treatment with Ca2+ chelators completely recovered Cd-induced cell viability and inhibited Cd-induced apoptosis and autophagy. Treatment with N-acetyl-l-cysteine (NAC) suppressed Cd-induced antioxidant enzyme levels, apoptosis, and autophagy. Collectively, Cd-induced oxidative stress triggers ER stress, leading to Ca2+-dependent calpain activation and subsequent activation of autophagy and apoptosis, resulting in immune suppression.

MAPK/JNK1 activation protects cells against cadmium-induced autophagic cell death via differential regulation of catalase and heme oxygenase-1 in oral cancer cells.

Antioxidant enzymes are related to oral diseases. We investigated the roles of heme oxygenase-1 (HO-1) and catalase in cadmium (Cd)-induced oxidative stress and the underlying molecular mechanism in oral cancer cells. Exposing YD8 cells to Cd reduced the expression levels of catalase and superoxide dismutase 1/2 and induced the expression of HO-1 as well as autophagy and apoptosis, which were reversed by N-acetyl-l-cysteine (NAC). Cd-exposed YD10B cells exhibited milder effects than YD8 cells, indicating that Cd sensitivity is associated with antioxidant enzymes and autophagy. Autophagy inhibition via pharmacologic and genetic modulations enhanced Cd-induced HO-1 expression, caspase-3 cleavage, and the production of reactive oxygen species (ROS). Ho-1 knockdown increased autophagy and apoptosis. Hemin treatment partially suppressed Cd-induced ROS production and apoptosis, but enhanced autophagy and CHOP expression, indicating that autophagy induction is associated with cellular stress. Catalase inhibition by pharmacological and genetic modulations increased Cd-induced ROS production, autophagy, and apoptosis, but suppressed HO-1, indicating that catalase is required for HO-1 induction. p38 inhibition upregulated Cd-induced phospho-JNK and catalase, but suppressed HO-1, autophagy, apoptosis. JNK suppression exhibited contrary results, enhancing the expression of phospho-p38. Co-suppression of p38 and JNK1 failed to upregulate catalase and procaspase-3, which were upregulated by JNK1 overexpression. Overall, the balance between the responses of p38 and JNK activation to Cd appears to have an important role in maintaining cellular homeostasis via the regulation of antioxidant enzymes and autophagy induction. In addition, the upregulation of catalase by JNK1 activation can play a critical role in cell protection against Cd-induced oxidative stress.

Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells.

BACKGROUND: Shikonin, a natural naphthoquinone pigment purified from Lithospermum erythrorhizon, induces necroptosis in various cancer types, but the mechanisms underlying the anticancer activity of shikonin in lung cancer are not fully understood. This study was designed to clarify whether shikonin causes necroptosis in non-small cell lung cancer (NSCLC) cells and to investigate the mechanism of action. METHODS: Multiplex and caspase 8 assays were used to analyze effect of shikonin on A549 cells. Cytometry with annexin V/PI staining and MTT assays were used to analyze the mode of cell death. Western blotting was used to determine the effect of shikonin-induced necroptosis and autophagy. Xenograft and orthotopic models with A549 cells were used to evaluate the anti-tumor effect of shikonin in vivo. RESULTS: Most of the cell death induced by shikonin could be rescued by the specific necroptosis inhibitor necrostatin-1, but not by the general caspase inhibitor Z-VAD-FMK. Tumor growth was significantly lower in animals treated with shikonin than in the control group. Shikonin also increased RIP1 protein expression in tumor tissues. Autophagy inhibitors, including methyladenine (3-MA), ATG5 siRNA, and bafilomycin A, enhanced shikonin-induced necroptosis, whereas RIP1 siRNA had no effect on the apoptotic potential of shikonin. CONCLUSIONS: Our data indicated that shikonin treatment induced necroptosis and autophagy in NSCLC cells. In addition, the inhibition of shikonin-induced autophagy enhanced necroptosis, suggesting that shikonin could be a novel therapeutic strategy against NSCLC.

Antinociceptive effect of intrathecal sec-O-glucosylhamaudol on the formalin-induced pain in rats.

BACKGROUND: The root of Peucedanum japonicum Thunb., a perennial herb found in Japan, the Philippines, China, and Korea, is used as an analgesic. In a previous study, sec-O-glucosylhamaudol (SOG) showed an analgesic effect. This study was performed to examine the antinociceptive effect of intrathecal SOG in the formalin test. METHODS: Male Sprague-Dawley rats were implanted with an intrathecal catheter. Rats were randomly treated with a vehicle and SOG (10 µg, 30 µg, 60 µg, and 100 µg) before formalin injection. Five percent formalin was injected into the hind-paw, and a biphasic reaction followed, consisting of flinching and licking behaviors (phase 1, 0-10 min; phase 2, 10-60 min). Naloxone was injected 10 min before administration of SOG 100 µg to evaluate the involvement of SOG with an opioid receptor. Dose-responsiveness and ED50 values were calculated. RESULTS: Intrathecal SOG showed a significant reduction of the flinching responses at both phases in a dose-dependent manner. Significant effects were showed from the dose of 30 µg and maximum effects were achieved at a dose of 100 µg in both phases. The ED50 value (95% confidence intervals) of intrathecal SOG was 30.3 (25.8-35.5) µg during phase 1, and 48.0 (41.4-55.7) during phase 2. The antinociceptive effects of SOG (100 µg) were significantly reverted at both phases of the formalin test by naloxone. CONCLUSIONS: These results demonstrate that intrathecal SOG has a very strong antinociceptive effect in the formalin test and it seems the effect is related to an opioid receptor.

Activation of AMPK by berberine induces hepatic lipid accumulation by upregulation of fatty acid translocase CD36 in mice.

Emerging evidence has shown that berberine has a protective effect against metabolic syndrome such as obesity and type II diabetes mellitus by activating AMP-activated protein kinase (AMPK). AMPK induces CD36 trafficking to the sarcolemma for fatty acid uptake and oxidation in contracting muscle. However, little is known about the effects of AMPK on CD36 regulation in the liver. We investigated whether AMPK activation by berberine affects CD36 expression and fatty acid uptake in hepatocytes and whether it is linked to hepatic lipid accumulation. Activation of AMPK by berberine or transduction with adenoviral vectors encoding constitutively active AMPK in HepG2 and mouse primary hepatocytes increased the expression and membrane translocation of CD36, resulting in enhanced fatty acid uptake and lipid accumulation as determined by BODIPY-C16 and Nile red fluorescence, respectively. Activation of AMPK by berberine induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and subsequently induced CCAAT/enhancer-binding protein ß (C/EBPß) binding to the C/EBP-response element in the CD36 promoter in hepatocytes. In addition, hepatic CD36 expression and triglyceride levels were increased in normal diet-fed mice treated with berberine, but completely prevented when hepatic CD36 was silenced with adenovirus containing CD36-specific shRNA. Taken together, prolonged activation of AMPK by berberine increased CD36 expression in hepatocytes, resulting in fatty acid uptake via processes linked to hepatocellular lipid accumulation and fatty liver.