PLIN2 promotes HCC cells proliferation by inhibiting the degradation of HIF1α.

PLIN2 has been found to be dysregulated in several human malignancies, which influences cancer progression. However, the roles of PLIN2 in regulating hepatocellular carcinoma (HCC) progression are still unclear. Here, we revealed that PLIN2 was frequently upregulated in HCC cells and tissues, and increased PLIN2 expression was associated with poor prognosis outcomes in HCC. In HCC cells, overexpressing PLIN2 promoted cell proliferation, PLIN2-deficiency inhibited cell vitality. Mechanistically, silencing of PLIN2 expression downregulated hypoxia inducible factor 1-α (HIF1α) expression and this downregulation in turn inhibited the targeting genes of HIF1α. Furthermore, we found that PLIN2 stabilized and retarded the degradation of the HIF1α through autophagy-lysosomal pathway by inhibiting AMPK/ULK1. Collectively, we clarified the carcinogenic role of PLIN2 in HCC and suggested a prognostic biomarker for diagnosis and clinical therapy in the future.

Silibinin relieves UVB-induced apoptosis of human skin cells by inhibiting the YAP-p73 pathway.

Excessive exposure to UVB induces skin diseases. Silibinin, a flavonolignan used for treating liver diseases, is found to be effective against UVB-caused skin epidermal and dermal cell damage. In this study we investigated the molecular mechanisms underlying. Human nonmalignant immortalized keratinocyte HaCaT cells and neonatal human foreskin fibroblasts HFFs were exposed to UVB irradiation. We showed that pre-treatment with silibinin dose-dependently decreased UVB-induced apoptosis of HaCaT cells. Furthermore, we showed that silibinin treatment inhibited nuclear translocation of YAP after UVB irradiation. Molecular docking analysis and DARTS assay confirmed the direct interaction of silibinin with YAP. Silencing YAP by siRNA had no influence on the survival of HaCaT cells, whereas inhibiting classical YAP-TEAD signaling pathway by siRNA targeting TEAD1 or its pharmaceutical inhibitor verteporfin further augmented UVB-induced apoptosis, suggesting that YAP-TEAD pathway was prosurvival, which did not participate in the protective effect of silibinin. We then explored the pro-apoptotic YAP-p73 pathway. p73 was upregulated in UVB-irradiated cells, but reduced by silibinin cotreatment. The mRNA and protein levels of p73 target genes (PML, p21 and Bax) were all increased by UVB but decreased by silibinin co-treatment. Inhibiting p73 by using siRNA reduced UVB-induced apoptosis, suggesting that downregulation of p73 was responsible for the cytoprotective effect of silibinin. In HFFs, the upregulated YAP-p73 pathway by UVB irradiation was also suppressed by silibinin. Collectively, YAP-p73 pathway is a major cause of the death of UVB-exposed epidermal HaCaT cells and dermal HFFs. Silibinin directly inhibits YAP-p73 pathway, exerting the protective action on UVB-irradiated skin cells.

AQP3-mediated H2 O2 uptake inhibits LUAD autophagy by inactivating PTEN.

It is widely accepted that redox reprogramming participates in malignant transformation of lung adenocarcinoma (LUAD). However, the source of excessive reactive oxygen species (ROS) and the downstream signaling regulatory mechanism are complicated and unintelligible. In the current study, we newly identified the aquaporin 3 (AQP3) as a LUAD oncogenic factor with capacity to transport exogenous hydrogen peroxide (H2 O2 ) and increase intracellular ROS levels. Subsequently, we demonstrated that AQP3 was necessary for the facilitated diffusion of exogenous H2 O2 in LUAD cells and that the AQP3-dependent transport of H2 O2 accelerated cell growth and inhibited rapamycin-induced autophagy. Mechanistically, AQP3-mediated H2 O2 uptake increased intracellular ROS levels to inactivate PTEN and activate the AKT/mTOR pathway to subsequently inhibit autophagy and promote proliferation in LUAD cells. Finally, we suggested that AQP3 depletion retarded subcutaneous tumorigenesis in vivo and simultaneously decreased ROS levels and promoted autophagy. These findings underscore the importance of AQP3-induced oxidative stress in malignant transformation and suggest a therapeutic target for LUAD.

Interference of silibinin with IGF-1R signalling pathways protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis.

Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis.

The roles of E3 ubiquitin ligases in cancer progression and targeted therapy.

Ubiquitination is one of the most important post-translational modifications which plays a significant role in conserving the homeostasis of cellular proteins. In the ubiquitination process, ubiquitin is conjugated to target protein substrates for degradation, translocation or activation, dysregulation of which is linked to several diseases including various types of cancers. E3 ubiquitin ligases are regarded as the most influential ubiquitin enzyme owing to their ability to select, bind and recruit target substrates for ubiquitination. In particular, E3 ligases are pivotal in the cancer hallmarks pathways where they serve as tumour promoters or suppressors. The specificity of E3 ligases coupled with their implication in cancer hallmarks engendered the development of compounds that specifically target E3 ligases for cancer therapy. In this review, we highlight the role of E3 ligases in cancer hallmarks such as sustained proliferation via cell cycle progression, immune evasion and tumour promoting inflammation, and in the evasion of apoptosis. In addition, we summarise the application and the role of small compounds that target E3 ligases for cancer treatment along with the significance of targeting E3 ligases as potential cancer therapy.

GPR35 antagonist CID-2745687 attenuates anchorage-independent cell growth by inhibiting YAP/TAZ activity in colorectal cancer cells.

Background and purpose: GPR35, a member of the orphan G-protein-coupled receptor, was recently implicated in colorectal cancer (CRC). However, whether targeting GPR35 by antagonists can inhibit its pro-cancer role has yet to be answered. Experimental approach: We applied antagonist CID-2745687 (CID) in established GPR35 overexpressing and knock-down CRC cell lines to understand its anti-cell proliferation property and the underlying mechanism. Key results: Although GPR35 did not promote cell proliferation in 2D conditions, it promoted anchorage-independent growth in soft-agar, which was reduced by GPR35 knock-down and CID treatment. Furthermore, YAP/TAZ target genes were expressed relatively higher in GPR35 overexpressed cells and lower in GPR35 knock-down cells. YAP/TAZ activity is required for anchorage-independent growth of CRC cells. By detecting YAP/TAZ target genes, performing TEAD4 luciferase reporter assay, and examining YAP phosphorylation and TAZ protein expression level, we found YAP/TAZ activity is positively correlated to GPR35 expression level, which CID disrupted in GPR35 overexpressed cells, but not in GPR35 knock-down cells. Intriguingly, GPR35 agonists did not promote YAP/TAZ activity but ameliorated CID's inhibitory effect; GPR35-promoted YAP/TAZ activity was only partly attenuated by ROCK1/2 inhibitor. Conclusion and implications: GPR35 promoted YAP/TAZ activity partly through Rho-GTPase with its agonist-independent constitutive activity, and CID exhibited its inhibitory effect. GPR35 antagonists are promising anti-cancer agents that target hyperactivation and overexpression of YAP/TAZ in CRC.

Aminosalicylates target GPR35, partly contributing to the prevention of DSS-induced colitis.

GPR35, a class A G-protein-coupled receptor, is considered an orphan receptor; the endogenous ligand and precise physiological function of GPR35 remain obscure. GPR35 is expressed relatively highly in the gastrointestinal tract and immune cells. It plays a role in colorectal diseases like inflammatory bowel diseases (IBDs) and colon cancer. More recently, the development of GPR35 targeting anti-IBD drugs is in solid request. Nevertheless, the development process is in stagnation due to the lack of a highly potent GPR35 agonist that is also active comparably in both human and mouse orthologs. Therefore, we proposed to find compounds for GPR35 agonist development, especially for the human ortholog of GPR35. As an efficient way to pick up a safe and effective GPR35 targeting anti-IBD drug, we screened Food and Drug Administration (FDA)-approved 1850 drugs using a two-step DMR assay. Interestingly, we found aminosalicylates, first-line medicine for IBDs whose precise target remains unknown, exhibited activity on both human and mouse GPR35. Among these, pro-drug olsalazine showed the most potency on GPR35 agonism, inducing ERK phosphorylation and ß-arrestin2 translocation. In dextran sodium sulfate (DSS)-induced colitis, the protective effect on disease progression and inhibitory effect on TNFα mRNA expression, NF-κB and JAK-STAT3 pathway of olsalazine are compromised in GPR35 knock-out mice. The present study identified a target for first-line medicine aminosalicylates, highlighted that uncleaved pro-drug olsalazine is effective, and provided a new concept for the design of aminosalicylic GPR35 targeting anti-IBD drug.

Multidirectional characterization of cellular composition and spatial architecture in human multiple primary lung cancers.

Multiple primary lung cancers (MPLCs) pose diagnostic and therapeutic challenges in clinic. Here, we orchestrated the cellular and spatial architecture of MPLCs by combining single-cell RNA-sequencing and spatial transcriptomics. Notably, we identified a previously undescribed sub-population of epithelial cells termed as CLDN2+ alveolar type II (AT2) which was specifically enriched in MPLCs. This subtype was observed to possess a relatively stationary state, play a critical role in cellular communication, aggregate spatially in tumor tissues, and dominate the malignant histopathological patterns. The CLDN2 protein expression can help distinguish MPLCs from intrapulmonary metastasis and solitary lung cancer. Moreover, a cell surface receptor-TNFRSF18/GITR was highly expressed in T cells of MPLCs, suggesting TNFRSF18 as one potential immunotherapeutic target in MPLCs. Meanwhile, high inter-lesion heterogeneity was observed in MPLCs. These findings will provide insights into diagnostic biomarkers and therapeutic targets and advance our understanding of the cellular and spatial architecture of MPLCs.

Midkine noncanonically suppresses AMPK activation through disrupting the LKB1-STRAD-Mo25 complex.

Midkine (MDK), a secreted growth factor, regulates signal transduction and cancer progression by interacting with receptors, and it can be internalized into the cytoplasm by endocytosis. However, its intracellular function and signaling regulation remain unclear. Here, we show that intracellular MDK interacts with LKB1 and STRAD to disrupt the LKB1-STRAD-Mo25 complex. Consequently, MDK decreases the activity of LKB1 to dampen both the basal and stress-induced activation of AMPK by glucose starvation or treatment of 2-DG. We also found that MDK accelerates cancer cell proliferation by inhibiting the activation of the LKB1-AMPK axis. In human cancers, compared to other well-known growth factors, MDK expression is most significantly upregulated in cancers, especially in liver, kidney and breast cancers, correlating with clinical outcomes and inversely correlating with phosphorylated AMPK levels. Our study elucidates an inhibitory mechanism for AMPK activation, which is mediated by the intracellular MDK through disrupting the LKB1-STRAD-Mo25 complex.

Norcantharidin overcomes vemurafenib resistance in melanoma by inhibiting pentose phosphate pathway and lipogenesis via downregulating the mTOR pathway.

Melanoma is the most aggressive type of skin cancer with a high incidence and low survival rate. More than half of melanomas present the activating BRAF mutations, along which V600E mutant represents 70%-90%. Vemurafenib (Vem) is an FDA-approved small-molecule kinase inhibitor that selectively targets activated BRAF V600E and inhibits its activity. However, the majority of patients treated with Vem develop acquired resistance. Hence, this study aims to explore a new treatment strategy to overcome the Vem resistance. Here, we found that a potential anticancer drug norcantharidin (NCTD) displayed a more significant proliferation inhibitory effect against Vem-resistant melanoma cells (A375R) than the parental melanoma cells (A375), which promised to be a therapeutic agent against BRAF V600E-mutated and acquired Vem-resistant melanoma. The metabolomics analysis showed that NCTD could, especially reverse the upregulation of pentose phosphate pathway and lipogenesis resulting from the Vem resistance. In addition, the transcriptomic analysis showed a dramatical downregulation in genes related to lipid metabolism and mammalian target of the rapamycin (mTOR) signaling pathway in A375R cells, but not in A375 cells, upon NCTD treatment. Moreover, NCTD upregulated butyrophilin (BTN) family genes, which played important roles in modulating T-cell response. Consistently, we found that Vem resistance led to an obvious elevation of the p-mTOR expression, which could be remarkably reduced by NCTD treatment. Taken together, NCTD may serve as a promising therapeutic option to resolve the problem of Vem resistance and to improve patient outcomes by combining with immunomodulatory therapy.

USP22 regulates lipidome accumulation by stabilizing PPARγ in hepatocellular carcinoma.

Elevated de novo lipogenesis is considered to be a crucial factor in hepatocellular carcinoma (HCC) development. Herein, we identify ubiquitin-specific protease 22 (USP22) as a key regulator for de novo fatty acid synthesis, which directly interacts with deubiquitinates and stabilizes peroxisome proliferator-activated receptor gamma (PPARγ) through K48-linked deubiquitination, and in turn, this stabilization increases acetyl-CoA carboxylase (ACC) and ATP citrate lyase (ACLY) expressions. In addition, we find that USP22 promotes de novo fatty acid synthesis and contributes to HCC tumorigenesis, however, this tumorigenicity is suppressed by inhibiting the expression of PPARγ, ACLY, or ACC in in vivo tumorigenesis experiments. In HCC, high expression of USP22 positively correlates with PPARγ, ACLY or ACC expression, and associates with a poor prognosis. Taken together, we identify a USP22-regulated lipogenesis mechanism that involves the PPARγ-ACLY/ACC axis in HCC tumorigenesis and provide a rationale for therapeutic targeting of lipogenesis via USP22 inhibition.

Silibinin treatment protects human skin cells from UVB injury through upregulation of estrogen receptors.

Ultraviolet B (UVB) from the sunlight is a major environmental cause for human skin damages, inducing cell death, inflammation, senescence and even carcinogenesis. The natural flavonoid silibinin, clinically used as liver protectant, has protective effects against UVB-caused skin injury in vivo and in vitro. Silibinin is often classified as a phytoestrogen, because it modulates the activation of estrogen receptors (ERs). However, whether silibinin's estrogenic effect contributes to the skin protection against UVB injury remains to be elucidated. The issue was explored in this study by using the human foreskin dermal fibroblasts (HFF) and human non-malignant immortalized keratinocytes (HaCaT). In HFF, pre-treatment with silibinin rescued UVB-irradiated cells from apoptosis. Interestingly, silibinin increased the whole cellular and nuclear levels of ERα and ERß in UVB-irradiated cells. Activation of ERs by treatment with estradiol elevated the cell survival and reduced apoptosis in UVB-treated cells. ERα agonist increased cell survival, while its antagonist decreased it. ERß agonist also increased cell survival, but the antagonist had no effect on cell survival. Transfection of the cells with the small interfering RNAs (si-RNAs) to ERα or ERß diminished the protective effect of silibinin on UVB-irradiated cells. In UVB-treated HaCaT cells, both ERα and ERß were increased by silibinin treatment. Inhibition of activation and expression of ERα or ERß by specific antagonists and si-RNAs, respectively, reduced cell survival in UVB-treated HaCaT cells regardless of silibinin treatment. Taken together, it is summarized that silibinin up-regulates both ERα and ERß pathways in UVB-treated dermal HFF cells and epidermal HaCaT cells, leading to protection of skin from UVB-damage.

YB1 regulates miR-205/200b-ZEB1 axis by inhibiting microRNA maturation in hepatocellular carcinoma.

BACKGROUND: Y-box binding protein 1 (YB1 or YBX1) plays a critical role in tumorigenesis and cancer progression. However, whether YB1 affects malignant transformation by modulating non-coding RNAs remains largely unknown. This study aimed to investigate the relationship between YB1 and microRNAs and reveal the underlying mechanism by which YB1 impacts on tumor malignancy via miRNAs-mediated regulatory network. METHODS: The biological functions of YB1 in hepatocellular carcinoma (HCC) cells were investigated by cell proliferation, wound healing, and transwell invasion assays. The miRNAs dysregulated by YB1 were screened by microarray analysis in HCC cell lines. The regulation of YB1 on miR-205 and miR-200b was determined by quantitative real-time PCR, dual-luciferase reporter assay, RNA immunoprecipitation, and pull-down assay. The relationships of YB1, DGCR8, Dicer, TUT4, and TUT1 were identified by pull-down and coimmunoprecipitation experiments. The cellular co-localization of YB1, DGCR8, and Dicer were detected by immunofluorescent staining. The in vivo effect of YB1 on tumor metastasis was determined by injecting MHCC97H cells transduced with YB1 shRNA or shControl via the tail vein in nude BALB/c mice. The expression levels of epithelial to mesenchymal transition markers were detected by immunoblotting and immunohistochemistry assays. RESULTS: YB1 promoted HCC cell migration and tumor metastasis by regulating miR-205/200b-ZEB1 axis partially in a Snail-independent manner. YB1 suppressed miR-205 and miR-200b maturation by interacting with the microprocessors DGCR8 and Dicer as well as TUT4 and TUT1 via the conserved cold shock domain. Subsequently, the downregulation of miR-205 and miR-200b enhanced ZEB1 expression, thus leading to increased cell migration and invasion. Furthermore, statistical analyses on gene expression data from HCC and normal liver tissues showed that YB1 expression was positively associated with ZEB1 expression and remarkably correlated with clinical prognosis. CONCLUSION: This study reveals a previously undescribed mechanism by which YB1 promotes cancer progression by regulating the miR-205/200b-ZEB1 axis in HCC cells. Furthermore, these results highlight that YB1 may play biological functions via miRNAs-mediated gene regulation, and it can serve as a potential therapeutic target in human cancers.

Identification and Characterization of Robust Hepatocellular Carcinoma Prognostic Subtypes Based on an Integrative Metabolite-Protein Interaction Network.

Metabolite-protein interactions (MPIs) play key roles in cancer metabolism. However, our current knowledge about MPIs in cancers remains limited due to the complexity of cancer cells. Herein, the authors construct an integrative MPI network and propose a MPI network based hepatocellular carcinoma (HCC) subtyping and mechanism exploration workflow. Based on the expressions of hub proteins on the MPI network, two prognosis-distinctive HCC subtypes are identified. Meanwhile, multiple interdependent features of the poor prognostic subtype are observed, including hypoxia, DNA hypermethylation of metabolic pathways, fatty acid accumulation, immune pathway up-regulation, and exhausted T-cell infiltration. Notably, the immune pathway up-regulation is probably induced by accumulated unsaturated fatty acids which are predicted to interact with multiple immune regulators like SRC and TGFB1. Moreover, based on tumor microenvironment compositions, the poor prognostic subtype is further divided into two sub-populations showing remarkable differences in metabolism. The subtyping shows a strong consistency across multiple HCC cohorts including early-stage HCC. Overall, the authors redefine robust HCC prognosis subtypes and identify potential MPIs linking metabolism to immune regulations, thus promoting understanding and clinical applications about HCC metabolism heterogeneity.

F-box proteins and cancer: an update from functional and regulatory mechanism to therapeutic clinical prospects.

E3 ubiquitin ligases play a critical role in cellular mechanisms and cancer progression. F-box protein is the core component of the SKP1-cullin 1-F-box (SCF)-type E3 ubiquitin ligase and directly binds to substrates by various specific domains. According to the specific domains, F-box proteins are further classified into three sub-families: 1) F-box with leucine rich amino acid repeats (FBXL); 2) F-box with WD 40 amino acid repeats (FBXW); 3) F-box only with uncharacterized domains (FBXO). Here, we summarize the substrates of F-box proteins, discuss the important molecular mechanism and emerging role of F-box proteins especially from the perspective of cancer development and progression. These findings will shed new light on malignant tumor progression mechanisms, and suggest the potential role of F-box proteins as cancer biomarkers and therapeutic targets for future cancer treatment.

Activated toll-like receptor 4 is involved in oridonin-induced phagocytosis via promotion of migration and autophagy-lysosome pathway in RAW264.7 macrophages.

In our previous study, we demonstrated that oridonin enhances phagocytosis of apoptotic bodies by macrophage-like cells by inducing autophagy. However, the direct sensor of autophagy and the key event controlling phagocytosis remains unknown. Herein, we showed that Toll-like receptor 4 (TLR4), known to mediate immune responses, was activated by oridonin. Activated TLR4 contributes to phagocytosis of apoptotic cells by RAW264.7 macrophages. Indeed, inhibition or small interfering RNA (siRNA) silencing of TLR4 significantly attenuated oridonin-induced phagocytosis. Inhibition of TLR4 also decreased the level of autophagy and its associated proteins, Beclin-1 and light chain 3 (LC3), suggesting that activated TLR4 is involved in activation of autophagy. LPS-induced activation of TLR4 promoted phagocytosis and autophagy progression. Activation of TLR4 accompany increase in activities of lysosome acid phosphatase and cathepsin B as well as in up-regulation of lysosomal-associated membrane protein (LAMP 1 and 2) levels. Furthermore, TLR4 in association with translocation to cytoplasm leads to macrophage motility or migration through increased plasticity of skeleton and/or membrane structure. These results suggest that oridonin-induced phagocytosis of apoptotic bodies by macrophages is TLR4 signal pathway-mediated, via activation of the autophagy-lysosome pathway as well as increase of cell migration.

Persistent IKKα phosphorylation induced apoptosis in UVB and Poly I:C co-treated HaCaT cells plausibly through pro-apoptotic p73 and abrogation of IκBα.

Toll-like receptor 3 (TLR3), a member of pattern recognition receptors, is reported to initiate skin inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. Recently, we have discovered the NF-κB pathway activated by TLR3 is involved in apoptosis of UVB-Poly I:C-treated HaCaT cells. The real culprit for apoptosis has not been precisely identified since the system of NF-κB pathway is complex. In this study, we silenced main transcriptional factors in NF-κB family, RelA, RelB and c-Rel, but to our surprise the results show that none of them participate in apoptosis induction in UVB-Poly I:C-treated HaCaT cells. Therefore, we moved to investigate the apoptosis-associated molecules in the upstream of NF-κB pathway. We firstly checked the expression of IκBα, an NF-κB inhibitor. UVB (4.8 mJ/cm2) and Poly I:C (0.3 µg/mL) co-treatment decreased IκBα expression level in a time-dependent manner. Silencing IκBα with siRNA further enhanced UVB-Poly I:C-induced cell death. We then investigated IκB kinase (IKK) complex that contributes to the degradation of IκBα. IKK is composed of IKKα, IKKß and NEMO. Treatment with IKK-16, an IKKα/ß inhibitor, significantly diminished UVB-Poly I:C-induced IκBα degradation and thus apoptosis. Silencing either IKKα or NEMO but not IKKß with corresponding siRNA inhibited apoptosis. Tumor repressor p73, a homologue of p53, is reported to mediate IKKα-induced apoptosis in DNA damage response. Silencing p73 reduced cell apoptosis in UVB-Poly I:C-treated HaCaT cells. In summary, UVB and Poly I:C co-treatment activates IKKα and NEMO, which diminishes anti-apoptotic IκBα, resulting in enhancement of apoptosis through p73. The findings partially clarify the possible molecular mechanism of pro-apoptotic NF-κB pathway activated by TLR3 in the fate of UVB-irradiated epidermis.

Type I collagen promotes primary cilia growth through down-regulating HDAC6-mediated autophagy in confluent mouse embryo fibroblast 3T3-L1 cells.

Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abnormal ciliogenesis and cilia length are suggested to be associated with hypertension and obesity as well as diseases such as Meckel-Gruber syndrome. Extracellular matrix (ECM), comprising cellular microenvironment, influences cell shape and proliferation. However, influence of ECM on cilia biogenesis has not been well studied. In this study we examined the effects of type I collagen (col I), the major component of ECM, on primary cilia growth. When cultured on collagen-coated dishes, confluent 3T3-L1 cells were found to exhibit fibroblast-like morphology, which was different from the cobblestone-like shape on non-coated dishes. The level of autophagy in the cells cultured on col I-coated dishes was attenuated compared with the cells cultured on non-coated dishes. The cilia of the cells cultured on col I-coated dishes became longer, accompanying increased expression of essential proteins for cilia assembly. Transfection of the siRNA targeting microtubule-associated protein light chain 3 (LC3) further enhanced the length of primary cilia, suggesting that col I positively regulated cilia growth through inhibition of autophagy. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy in our previous study on primary cilia, was down-regulated with col I. 3T3-L1 cells treated with the siRNA against HDAC6 reduced the autophagy level and enhanced collagen-induced cilia elongation, implying that HDAC6 was involved in mediating autophagy. In conclusion, col I promotes cilia growth through repressing the HDAC-autophagy pathway that can be involved in the interaction between primary cilia and col I.

Sub-lethal ultraviolet B irradiation and Poly I:C treatment synergistically induced apoptosis of HaCaT cells through NF-κB pathway.

Ultraviolet B (UVB) irradiation exerts multiple effects on skin cells, inducing apoptosis, senescence and carcinogenesis. Toll-like receptor 3, a member of pattern recognition receptors, is reported to initiate inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. It has not been studied, however, whether apoptosis induction in UVB irradiation is attributed to TLR3 activation. Here, we report on the pro-apoptotic role of TLR3 in UVB-irradiated epidermal cells. Poly I:C, an analogue of dsRNA that activates TLR3, was used in combination with sub-lethal UVB (4.8 mJ/cm2) irradiation for investigating the effects of TLR3 activation on human immortalized keratinocyte HaCaT cells. Although sub-lethal dose of either Poly I:C or UVB alone did not induce cell death, UVB-Poly I:C co-treatment synergistically induced cell death by activation of caspase-3 and cleavages of ICAD and PARP, with apoptotic features when stained with Annexin V/PI or Hoechst 33342. Treatment with pan-caspase inhibitor, Z-VAD, attenuated UVB-Poly I:C-induced cell death. Silencing TLR3 by siRNA rescued HaCaT cells from UVB-Poly I:C-induced apoptosis. NF-κB, a major downstream component of TLR3 pathway, that usually negatively regulates the classical TLR3 apoptotic pathway, was analyzed by western blotting and immunofluorescence confocal microscopy. The results indicate to our surprise that NF-κB is translocated to nucleus in the cells co-treated with UVB-Poly I:C. The nuclear translocation of NF-κB is attenuated by TLR3 silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-Poly I:C-induced apoptosis. Therefore, we conclude that NF-κB pathway plays a cytotoxic role in UVB-Poly I:C-treated HaCaT cells, mediating TLR3-related apoptosis.

Silibinin protects murine fibroblast L929 cells from UVB-induced apoptosis through the simultaneous inhibition of ATM-p53 pathway and autophagy.

Ultraviolet B (UVB) is a major cause of skin inflammation, leading to skin damage. Our previous in vivo study revealed that a natural flavonoid silibinin had marked anti-inflammatory effect on UVB-exposed murine skin. UVB exposure caused reduced autophagy in epidermis while it promoted autophagy in dermis. Nevertheless, silibinin inhibited the inflammatory flux in the skin epidermis as well as dermis through the modulation of autophagy. In order to elucidate the underlying protective mechanisms of silibinin for UVB damage on skin, separate studies on epidermis and dermis are helpful. Derived from the normal tissue of the mouse, L929 cells are capable of representing some characteristics of dermal cells. UVB irradiation caused L929 cell apoptosis in a time- and dose-dependent manner. Ataxia-telangiectasia-mutated (ATM) protein and p53 were activated to cause cell apoptosis, accompanying upregulation of the autophagic flux. The pharmacological inhibition of ATM, p53 and autophagy or the transfection with autophagy-associated protein-targeted small interfering RNAs showed that the UVB-activated ATM-p53 axis and autophagy formed a positive feedback loop, which synergistically promoted cell apoptosis. Silibinin treatment simultaneously repressed the activation of ATM-p53 and autophagy and thereby protected UVB-irradiated L929 cells from apoptotic death.