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Isoalantolactone (Iso) is a bioactive lactone isolated from the root of Inula helenium L, which has been reported to have many pharmacological effects. To investigate the role and mechanism of isoalantolactone in chronic myeloid leukemia (CML), we first investigated isoalantolactone's anti-proliferative effects on imatinib-sensitive and imatinib-resistant CML cells by CCK8. Flow cytometry was used to detect isoalantolactone-induced cell apoptosis. Survivin was overexpressed in KBM5 and KBM5T315I cells using the lentivirus vector pSIN-3×flag-PURO. In KBM5 and KBM5T315I cells, shRNA was used to knockdown survivin. Cellular Thermal Shift Assay (CETSA) was used to detect the interaction between isoalantolactone and survivin. The ubiquitin of survivin induced by isoalantolactone was detected through immunoprecipitation. Quantitative polymerase-chain reaction (Q-PCR) and western blotting were used to detect the levels of mRNA and protein. Isoalantolactone inhibits the proliferation and promotes apoptosis of imatinib-resistant CML cells. Although isoalantolactone inhibits the proteins of BCR-ABL and survivin, it cannot inhibit survivin and BCR-ABL mRNA levels. Simultaneously, it was shown that isoalantolactone can degrade survivin protein by increasing ubiquitination. It was demonstrated that isoalantolactone-induced survivin mediated downregulation of BCR-ABL protein. It was also revealed that isoalantolactone triggered BCR-ABL protein degradation via caspase-3. Altogether, isoalantolactone inhibits survivin through the ubiquitin proteasome pathway, and mediates BCR-ABL downregulation in a caspase-3 dependent manner. These data suggest that isoalantolactone is a natural compound, which can be used as a potential drug to treat TKI-resistant CML. [ABSTRACT FROM AUTHOR]

Abstract Background Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer. Methods Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice. Results Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression. Conclusion Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

MicroRNAs can act as tumour suppressors or oncogenes by regulating cellular differentiation, proliferation and apoptosis, and the dysregulation of miRNA is involved in the occurrence and development of NSCLC. Here, we provided evidence that miR‐92b as an oncogene in NSCLC by targeting PTEN/AKT. We found that miR‐92b was up‐regulated in human NSCLC tissues and cell lines. MiR‐92b knockdown suppressed the NSCLC cells proliferation and migration in both in vivo and in vitro models. Conversely, miR‐92b overexpression induced an aggressive phenotype. Moreover, miR‐92b‐mediated regulation of NSCLC cell proliferation and migration depended on binding to PTEN mRNA, which then led to the degradation of PTEN and activation of the downstream AKT signalling pathway. Overall, this study revealed the oncogenic roles of miR‐92b in NSCLC by targeting PTEN/AKT, and provided novel insights for future treatments of NSCLC patients. Significance of the study: MiR‐92b was up‐regulated in human NSCLC tissues and cell lines. Our study demonstrated that miR‐92b as an oncogene in NSCLC by targeting PTEN/AKT in both in vivo and in vitro models and provided novel insights for future treatments of NSCLC patients. [ABSTRACT FROM AUTHOR]

Tumor immune escape is an important part of tumorigenesis and development. Tumor cells can develop a variety of immunosuppressive mechanisms to combat tumor immunity. Exploring tumor cells that escape immune surveillance through the molecular mechanism of related immunosuppression in-depth is helpful to develop the treatment strategies of targeted tumor immune escape. The latest studies show that CD24 on the surface of tumor cells interacts with Siglec-10 on the surface of immune cells to promote the immune escape of tumor cells. It is necessary to comment on the molecular mechanism of inhibiting the activation of immune cells through the interaction between CD24 on tumor cells and Siglec-10 on immune cells, and a treatment strategy of tumors through targeting CD24 on the surface of tumor cells or Siglec-10 on immune cells. [ABSTRACT FROM AUTHOR]

Oridonin could induce NB (neuroblastoma) cells growth inhibition by inducing apoptosis and cell cycle arrest, and the molecular mechanisms behind the effects deserve to be further explored. Here, oridonin was confirmed to cause the reactivation of p53 (cellular tumor antigen p53) to promote the expression of a series of apoptosis‐ and cell cycle arrest‐related proteins for the biological effects. During the process, oridonin relied on the caspase activation to cleave p53‐induced Mdm2 (E3 ubiquitin‐protein ligase Mdm2) to generate Mdm2‐p60. The generation of Mdm2‐p60 stabilized p53, and resulted in p53 accumulation for p53 continuous activation. In our research, it was also found that the reactivation of p53 induced by oridonin was closely related with the generation of ROS (reactive oxygen species). Taken together, these findings explain that oridonin exerts its anticancer activity partially by targeting the Mdm2‐p53 axis in NB cells, which lay an experimental base for future research of exploring the effects and molecular mechanisms of oridonin. [ABSTRACT FROM AUTHOR]

Small ubiquitin-like modifier (SUMOylation) is a reversible post-translational modification, which plays important roles in numerous biological processes. SUMO could be covalently attached to target proteins in an isopeptide bond manner that occurs via a lysine ε-amino group on the target proteins and the glycine on SUMO C-terminus. This covalent binding could affect the subcellular localization and stability of target proteins. SUMO modification can be reversed by members of the Sentrin/SUMO-specific proteases (SENPs) family, which are highly evolutionarily conserved from yeast to human. SENP2, a member of the SENPs family, mainly plays a physiological function in the nucleus. SENP2 can promote maturity of the SUMO and deSUMOylate for single-SUMO modified or poly-SUMO modified proteins. SENP2 can affect the related biological processes through its peptidase activity or the amino terminal transcriptional repression domain. It plays important roles by inhibiting or activating some molecular functions. Therefore, the research achievements of SENP2 are reviewed in order to understand its related functions and the underlying molecular mechanisms and provide a clue for future research on SENP2. [ABSTRACT FROM AUTHOR]

Cyclin D1 and cyclin E1, as vital regulatory factors of G1-S phase cell cycle progression, are frequently constitutive expressed and associated with pathogenesis and tumorigenesis in most human cancers and they have been regarded as promising targets for cancer therapy. In this study, we established NVP-BEZ235, a potent dual kinase inhibitor, could induce neuroblastoma cells proliferation inhibition without apoptosis activation. Moreover, we showed NVP-BEZ235 could induce neuroblastoma cells arrested at G0/G1 phase accompanied with significant reduction of the cyclin D1 and E1 proteins in a dose dependent manner at nanomole concentration. Additionally we found that GSK3β was dephosphorylated and activated by NVP-BEZ235 and then triggered cyclin D1 and cyclin E1 degradation through ubiquitination proteasome pathway, based on the evidences that NVP-BEZ235 induced downregulation of cyclin D1 and cyclin E1 were obviously recovered by proteasome inhibitor and the blockade of GSK3β contributed to remarkable rescue of cyclin D1 and cyclin E1. Analogous results about its anti-proliferation effects and molecular mechanism were observed on neuroblastoma xenograft mouse model in vivo. Therefore, these results indicate that NVP-BEZ235-induced cyclin D1 and cyclin E1 degradation, which happened through activating GSK3β, and GSK3β-dependent down-regulation of cyclin D1 and cyclin E1 should be available for anticancer therapeutics. [ABSTRACT FROM PUBLISHER]

Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced. [ABSTRACT FROM AUTHOR]

Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the clinical significance of Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) during angiogenesis in non-small-cell lung carcinoma. JAK2, phospho-JAK2 (pJAK2), STAT3, and phospho-STAT3 (pSATA3) were observed in 40/68 (58.8%), 39/68 (57.4%), 49/68 (72.1%) and 40/68 (58.8%) of the cases. The high expression levels of molecules involved in the JAK2/STAT3 signaling pathway were associated with a decreased survival rate. Of the total number of cases, 73.5% were positive for VEGF and 80.9% for bFGF. Microvessel density (MVD), as determined by CD34 staining and morphology, was higher in NSCLC samples with high pJAK2 and pSTAT3 expression, and the patients with high MVD had poor survival status. In addition, the expression of pSTAT3 correlated with VEGF (r =0.593) and bFGF (r =0.519) (p <0.05). Inhibiting JAK2 and knocking down STAT3 both suppressed STAT3 activation and reduced the expression of VEGF and bFGF in A549 and NCI-H292 cells, demonstrating that STAT3 activation was associated with VEGF and bFGF expression in the two human lung carcinoma cell lines. Therefore, STAT3 may be a critical molecular target for powerful intervention in NSCLC anti-angiogenesis therapy. [Copyright &y& Elsevier]

An in vivo study on enhancing epirubicin hydrochloride (EPI) inhibition on tumor growth by ultrasound (US) was reported. Five-week-old male nude mice were used and HL-60 cells were s.c. (subcutaneous injection) inoculated in axilla of these mice. Six groups were designed and five consecutive treatments were applied to investigate the inhibition on tumor growth and body weight growth. US applied locally to the tumor resulted in a substantially increased drug uptake in tumor cells. The inhibition on tumor growth depended on the position of drug injection and phospholipid-based microbubble (PMB) application. Tumor growth rate under group 1 (PMB+US) was similar to that of blank control. The order of the inhibition on tumor volume growth was: group 4 (s.c. EPI+PMB+US) > group 5 intraperitoneal (i.p. EPI+PMB+US) > group 2 (i.p. EPI) > group 3 (s.c. EPI+US) > group 1 (PMB+US). Similar conclusion was obtained from experimental measurements of tumor weight change. The order of animal survival status for EPI administration groups was: group 4 > group 5 > group 2 > group 3. Chemotherapeutic drug inhibition on tumor growth could be enhanced by local US combined with PMB, which might provide a potential application for US-mediated chemotherapy. [ABSTRACT FROM AUTHOR]

Abstract: AML1-ETO fusion protein, a product of leukemia-related chromosomal translocation t(8;21), was reported to upregulate expression of connexin-43 (Cx43), a member of gap junction-constituted connexin family. However, its mechanism(s) remains unclear. By bioinformatic analysis, here we showed that there are two putative AML1-binding consensus sequences followed by two activated protein (AP)1 sites in the 5′-flanking region upstream to Cx43 gene. AML1-ETO could directly bind to these two AML1-binding sites in electrophoretic mobility shift assay, but luciferase reporter assay revealed that the AML1 binding sites were not indispensable for Cx43 induction by AML1-ETO protein. Conversely, AP1 sites exerted an important role in this event. In agreement, AML1-ETO overexpression in leukemic U937 cells activated c-Jun N-terminal kinase (JNK), while its specific inhibitor SP600125 effectively abrogated AML1-ETO-induced Cx43 expression, indicating that JNK signaling pathway contributes to AML1-ETO induced Cx43 expression. These results would shed new insights for understanding mechanisms of AML1-ETO-associated leukemogenesis. [Copyright &y& Elsevier]

Background: Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer.Methods: Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice.Results: Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression.Conclusion: Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment. [ABSTRACT FROM AUTHOR]

CDK2 forms a complex with cyclin A and cyclin E to promote the progress of cell cycle, but when cyclin A and cyclin E are dissociated from the complex and degraded by the ubiquitin proteasome pathway, the fate of the inactive CDK2 is unclear. In this study, we found that the inactive CDK2 protein was degraded by autophagy-lysosome pathway. In the classic model of G0/G1 phase arrest induced by serum starvation, we found that the mRNA level in CDK2 did not change but the protein level decreased. Subsequently, using PI3K and AKT inhibitors and gene knockout methods, it was found that CDK2 degradation was mediated by the inhibition of PI3Kα/AKTT308. In addition, P62/SQSTM1 was found to bind to the inactivated CDK2 protein to help it enter autophagy-lysosome degradation in a CTSB-dependent manner. Taken together, these results confirm that the PI3Kα/AKTT308 inhibition leads to degradation of CDK2 protein in the autophagy-lysosome pathway. These data reveal a new molecular mechanism of CDK2 protein degradation and provide a new strategy and method for regulating CDK2 protein. • Autophagy-lysosome pathway is involved in serum starvation-induced degradation of CDK2 protein. • The downregulation of CDK2 protein is mediated by the inhibition of PI3Kα/AKTT308. • P62/SQSTM1 mediates the autophagic degradation of CDK2 protein. [ABSTRACT FROM AUTHOR]

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer. [ABSTRACT FROM AUTHOR]

The distinction between chromophobe renal cell carcinoma (ChRCC), clear cell renal cell carcinoma (CRCC) and renal oncocytoma may cause a diagnostic dilemma. The usefulness of DOG1, cyclin D1, CK7, CD117 and vimentin in the differential diagnosis of these renal epithelial tumors was investigated. DOG1 was positive in ChRCC (32 of 32, 100%) and in renal oncocytoma (21 of 21, 100%). In contrast, DOG1 was absent in all CRCC (0 of 30). Cyclin D1 was positive in renal oncocytomas (17 of 21, 81%) but negative in the ChRCC (0/23) and CRCC (0 of 30). CK7 was positive in ChRCC (30 of 32, 94%), but was negative in oncocytoma (only scattered single positive cells), and was only focal positive in two cases of CRCC. CD117 was expressed in 88% of ChRCC (28 of 32), 86% of renal oncocytoma (18 of 21), and was negative in all CRCC (0 of 30). Twenty-six of the 30 cases of CRCC were positive (87%) for vimentin with prominent membrane staining patterns. All 23 chromophobe carcinomas were negative for vimentin and 15 of 21 oncocytomas demonstrated focal vimentin positivity, but less than 10%. The above results demonstrate that: (1) DOG1 was very sensitive and specific marker for distinguish ChRCC from CRCC; (2) Cyclin D1 was a useful marker to discriminate between ChRCC and renal oncocytoma; (3) CK7 and CD117 were useful markers to distinguish ChRCC from renal oncocytoma and CRCC. (4) Vimentin was helpful for distinguishing clear cell RCC from chromophobe and oncocytoma (87% of clear cell RCC positive, negative in chromophobe, only focally positive in oncocytoma). (5) CK8/18, CK19, CD10, β-catenin and E-cadherin could not be used to distinguish ChRCC from renal oncocytoma and CRCC. [ABSTRACT FROM AUTHOR]

Abstract: Recombinant human erythropoietin (rHuEPO) reduces serum insulin levels, increases insulin sensitivity, and reduces insulin resistance (IR). However, the mechanisms behind these effects are unclear. This study aimed to investigate the mechanism by which rHuEPO effects IR in 3T3L1 adipocytes. After treatment with different concentrations of rHuEPO, glucose consumption, and tumor necrosis factor (TNF-α), adiponectin, and leptin levels were assayed with a commercial enzyme-linked immunosorbent assays. Endogenous erythropoietin receptor (EPOR) expression was inhibited using small interfering RNA (siRNA). EPOR protein and mRNA expression was detected via immunofluorescence and real-time PCR analyses, respectively. The expression of pAKT/AKT and p-STAT5/STAT5 was determined via Western blot analysis. rHuEPO treatment improved glucose uptake, increased adiponectin levels, and reduced TNF-α and leptin levels in 3T3L1 adipocytes with dexamethasone-induced IR. Whereas EPOR protein and gene expression was absent in preadipocytes, it was observed in mature 3T3L1 adipocytes. However, the expression of EPOR in insulin resistant 3T3L1 adipocytes was significantly decreased (p <0.05). rHuEPO increased the expression of EPOR, and upregulated the expression of pAKT/AKT and pSTAT5/STAT5 in 3T3L1 adipocytes (p <0.05), which was blocked by siEPOR, the phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002, and a STAT5 inhibitor, respectively. In summary, rHuEPO reduced IR in adipocytes by increasing glucose uptake and improving the adipokine profile. rHuEPO-induced EPOR protein expression and subsequent induction of pAKT and pSTAT5 suggest that the EPO–EPOR system may play a role in glucose metabolism within adipocytes. [Copyright &y& Elsevier]

Background: CDK4 is highly expressed and associated with poor prognosis and decreased survival in advanced neuroblastoma (NB). Targeting CDK4 degradation presents a potentially promising therapeutic strategy compared to conventional CDK4 inhibitors. However, the autophagic degradation of the CDK4 protein and its anti-proliferation effect in NB cells has not been mentioned., Results: We identified autophagy as a new pathway for the degradation of CDK4. Firstly, autophagic degradation of CDK4 is critical for NVP-BEZ235-induced G0/G1 arrest, as demonstrated by the overexpression of CDK4, autophagy inhibition, and blockade of autophagy-related genes. Secondly, we present the first evidence that p62 binds to CDK4 and then enters the autophagy-lysosome to degrade CDK4 in a CTSB-dependent manner in NVP-BEZ235 treated NB cells. Similar results regarding the interaction between p62 and CDK4 were observed in the NVP-BEZ235 treated NB xenograft mouse model., Conclusions: Autophagic degradation of CDK4 plays a pivotal role in G0/G1 cell cycle arrest in NB cells treated with NVP-BEZ235.

Background: The incidence of malignant melanoma accounts for only approximately 5% of skin malignant tumors, however, it accounts for 75% of its mortality. Long-chain non-coding RNA (lncRNA) has a wide range of functional activities. Disorders of lncRNAs may lead to the occurrence and development of melanoma, and may also be related to immunotherapy., Methods: The transcriptomic data of primary and metastatic melanoma patients and 331 immune-related genes were downloaded from skin cutaneous melanoma (SKCM) in the The Cancer Genome Atlas (TCGA) database. On this basis, 460 immunologically relevant lncRNAs were identified by constructing a co-expression network of immunogenic genes and lncRNAs in primary and metastatic melanoma patients. Prognostic genes were screened using univariate Cox regression analysis. ROC analysis was performed to evaluate the robustness of the prognostic signature., Results: Univariate correlation analysis showed that only 3 of the 23 immune-related lncRNAs were at high risk and the rest were at low risk. Signatures of 7 immune-related lncRNAs were identified by multivariate correlation analysis. The clinical correlation analysis showed that the 7 immune-related lncRNAs were associated with the clinical stage of primary and metastatic melanoma. Principal component analysis (PCA) showed that only 7 immune-related lncRNA signals divided tumor patients into high-risk and low-risk groups, while the low-risk group was enriched in the immune system process M13664 and immune response M19817 sets. PPI interaction network analysis showed that 11 G protein-coupled receptors and 6 corresponding ligands in the 2 gene sets affected the tumor microenvironment and were negatively related to the risk of the 7 immune-related lncRNAs. The tumor microenvironment immune cell infiltration analysis also supported the finding that anti-tumor immunity in the low-risk group was stronger than in the high-risk group., Conclusions: These results indicate that characteristics of the 7 immune-related lncRNAs have prognostic value for melanoma patients and can be used as potential immunotherapy targets., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-21-1794). The authors have no conflicts of interest to declare., (2021 Annals of Translational Medicine. All rights reserved.)

The BCR-ABL fusion protein with strong tyrosine kinase activity is one of the molecular biological bases of leukemia. Imatinib (Gleevec), a specific targeted drug for the treatment of chronic myeloid leukemia (CML), was developed for inhibiting the kinase activity of the BCR-ABL fusion protein. Despite the positive clinical efficacy of imatinib, the proportion of imatinib resistance has gradually increased. The main reason for the resistance is a decrease in sensitivity to imatinib caused by mutation or amplification of the BCR-ABL gene. In response to this phenomenon, the new generation of tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL fusion protein was developed to solve the problem. However this strategy only selectively inhibits the tyrosine kinase activity of the BCR-ABL protein without eliminating the BCR-ABL protein, it does not fundamentally cure the BCR-ABL-positive leukemia patients. With the accumulation of the knowledge of cellular molecular biology, it has become possible to specifically eliminate certain proteins by cellular proteases in a specific way. Therefore, the therapeutic strategy to induce the degradation of the BCR-ABL fusion protein is superior to the strategy of inhibiting its activity. The protein degradation strategy is also a solution to the TKI resistance caused by different BCR-ABL gene point mutations. In order to provide possible exploration directions and clues for eliminating the BCR-ABL fusion protein in tumor cells, we summarize the significant molecules involved in the degradation pathway of the BCR-ABL protein, as well as the reported potent compounds that can target the BCR-ABL protein for degradation., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The progressive infiltration of immune cells is associated with the progression of melanoma. Specifically, Th17 cells in melanoma microenvironment have both antitumor and protumor effects. It is now necessary to understand the contradictory data associated with how Th17 cells play a role in melanoma. This review will summarize the current knowledge regarding the potential mechanisms that may be involved in the effects of Th17 cells in melanoma progression. Currently, since adoptive transferring Th17 cells has been successful in eradicating melanoma in mice, it offers promise for next-generation adoptive cell transfer, as ex vivo expanded stemness-like memory Th17 cells which are induced by distinct cytokines or pharmacologic reagents may be infused into melanoma patients to potentiate treatment outcome.

Ubiquitin-specific protease 2 (USP2) has a regulatory function in cell growth or death and is involved in the pathogenesis of various diseases. USP2 gene can generate 7 splicing variants through alternative splicing, and 5 variants respectively as USP2-201, USP2-202, USP2-204, USP2-205, USP2-206 can encode proteins. The influence of circadian rhythm, nutrition and androgen on specific signaling molecules or cytokines can regulate the alternative splicing of USP2. Specifically, PKC activator, IL-1β, TNF-α, PDGF-BB, TGF-β1 are all regulatory factors for USP2's alternative splicing. USP2-201 plays a crucial role in cell cycle progression, and is also of great significance in EGFR recycling. USP2-202 can activate apoptosis signaling pathway to participate in cell apoptosis, and USP2-204 can induce cell anti-virus reaction to decrease. In general, we collect and summarize the factors involved in the alternative splicing of USP2 in this review to further understand the mechanism behind the USP2's alternative splicing., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Hematopoietic cells are regulated by many transcriptional factors during their development, among them the Ikaros family is one of the most important representatives. They have characteristic conserved structural motifs, binding with DNA specific short sequences-containing key gene promoter or enhancer, to regulate their transcription activity. Meanwhile, the Ikaros family interact with other related transcriptional regulators to regulate the development and differentiation of hematopoietic cells. The structure of the Ikaros family, which belong to the zinc finger transcription factor, including Ikaros, Helios, Aiolos, Eos and Pegasus, are encoded by IKZF1-5 genes, respectively. They are master regulators of hematopoiesis, playing important roles in the occurrence, development and function of hematopoietic cells such as lymphocytes via individual and joint regulation. When working abnormalities, they are often related with the occurrence and development of the disease. In this review, the research achievements of the Ikaros family in recent years are summarized. On the one hand, it is helpful to understand the role and significance of this family in hematopoietic system; on the other hand, it provides the possible research direction for further research work.

DNA damage repair is a kind of cellular self-protection mechanism in which some relevant proteins are activated when DNA damage response occurs in order to maintain the intracellular function stability and structure integrity. Post-translational modifications (PTMs) of proteins can rapidly confer to them more complicated structure and sophisticated function by covalently combining different small molecules with target proteins, which in turn plays an important regulatory role in DNA damage repair. It was reported that heterogeneous nuclear ribonucleoprotein K (hnRNP K) could be involved in DNA damage repair process under the regulation of its many post-translational modifications, including methylation, ubiquitination, sumoylation and phosphorylation. Here, we reviewed molecular mechanisms of hnRNP K protein post-translational modifications and their role in DNA damage repair, which will promote our understanding of how hnRNP K participating in the repair process to maintain the normal operation of biological activities in the cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The aberrant activation of PI3K/Akt/mTOR signaling pathway plays an important role in the oncogenesis, prognosis and chemotherapy resistance of neuroblastoma. However, NVP-BEZ235, a potent dual PI3K and mTOR inhibitor have not shown beneficial effects on neuroblastoma especially in terms of apoptosis induction as a single agent. We therefore attempted to explore an effective combination regimen to enhance the anticancer activity of NVP-BEZ235. Interestingly, we found that oridonin, a natural biologically active compound extracted from the Chinese medicinal herb Rabdosia rubescens, combined with NVP-BEZ235 markedly induced apoptosis of neuroblastoma cells. Notably, the synergistic activation of the apoptotic pathway was accompanied with enhanced autophagy as evidenced by significant decreased p62 expression as well as upregulated conversion of LC3-II. Suppression of the Beclin-1, a core component of the autophagy machinery, by means of shRNA resulted in diminished synergistic antitumor effect. Furthermore, the co-treatment with oridonin and NVP-BEZ235 was also much more effective than either agent alone in inhibiting the growth of neuroblastoma xenografts and in inducing tumor cells apoptosis. Taken together, our results suggest that the combination of NVP-BEZ235 and oridonin is a novel and potential strategy for neuroblastoma therapy.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the hnRNP family, which exists in the nucleus and the cytoplasm simultaneously. It is a multifunctional protein that can participate in a variety of regulatory progressions of gene expression and signal transduction, such as chromatin remodeling, transcription, RNA alternative splicing and translation. hnRNP K not only directly binds to the kinases, but also recruits the associated factors regarding transcription, splicing and translation to control gene expression, and therefore, it serves as a docking platform for integrating transduction pathways to nucleic acid-directed processes. Numerous studies also show that abnormal expression of hnRNP K is closely associated with the tumor formation. This protein is overexpressed in numerous types of cancer and its aberrant cytoplasmic localization is also associated with a worse prognosis for patients. These results consistently indicate that hnRNP K has a key role in cancer progression. To understand the hnRNP K pathophysiological process in tumor disease, the previous research results regarding the association between hnRNP K and tumors were reviewed.

The overexpression or abnormal activation of cyclo‑oxygenase‑2 (COX‑2) has been reported in pancreatic cancer cells. NS‑398, a selective inhibitor of COX‑2, is unable to inhibit pancreatic cancer cell proliferation, as determined by a Cell Counting Kit 8 assay. However, it does increase cancer cell invasiveness, and therefore the invasiveness of the PANC‑1 cells was determined, along with the activation of P38, which was assessed by western blotting. In the present study, to evaluate the mechanisms underlying the action of NS‑398 in pancreatic cancer cells, PANC‑1 cells were treated with NS‑398, and the invasion signaling pathways of cluster of differentiation (CD)147‑matrix metalloproteinase (MMP)‑2 and mitogen‑activated protein kinases were evaluated. The results showed that NS‑398‑induced the expression of CD147 and MMP‑2 via the activation of P38, which was involved in antiproliferative activity and induced pancreatic cancer cell invasiveness. The PANC‑1 cells were also co‑treated with CD147 small interfering (si)RNA and NS‑398, and it was found that the NS‑398‑induced activation of P38 was not inhibited by CD147 siRNA, however, the expression of MMP‑2 was inhibited. CD147 siRNA inhibited the invasiveness of the pancreatic cancer cells induced by NS‑398, but also restored NS‑398‑induced antiproliferative activity. These data indicated that P38 in the pancreatic cancer cells was non‑specifically activated by NS‑398. This activation induced the expression of CD147‑MMP‑2, opposed the antiproliferative activity of NS‑398 and increased the invasiveness of the PANC‑1 cells.

Gastric cancer cell are not particularly sensitive to Ara-C, a deoxycytidine analog that affects DNA synthesis. In the present study, AGS and MKN-45 gastric cancer cell lines were treated with Ara-C to determine its role in cell prolife-ration and apoptosis. The antiproliferative effect of Ara-C was assessed using the Cell Counting kit-8. Gelatinase zymography was utilized to detect the activity of MMP-2 and MMP-9, and an in vitro invasion assay was performed. Using RT-PCR, CD-147, MMP-2 and MPP-9 mRNA levels were assessed in AGS cells with various doses of Ara-C treatment. CD-147, MMP-2 and MMP-9 protein levels were analysed in Ara-C‑treated AGS and MKN-45 cells. AGS cells were treated with or without U-0126 or siRNA-CD147 and/or Ara-C for 24 h, and an in vitro invasion assay was performed. Although low-dose Ara-C had no obvious effect on cell proliferation, it upregulated the expression of MMP-2, MMP-9 and CD-147 and ERK activation. Low-dose Ara-C increased gastric cancer cell invasion. U-0126 and siRNA-CD-147 inhibited the induction of Ara-C in gastric cancer cell invasion. Therefore, Ara-C enhances the invasiveness of gastric cancer cells by expression of CD-147 /MMP-2 and MMP-9 via the ERK signaling pathway. The results are therefore useful in the prevention of Ara-C collateral damage associated with standard, conventional protocols of chemotherapy administration.

The expression of miR-145 with tumor suppressor function is decreased in lung cancer cells. Epidermal growth factor receptor (EGFR) signaling pathway is abnormally activated in lung cancer cells. It is not clear whether the EGFR signaling pathway is involved in the regulation of miR-145 expression in lung cancer. In the present study, we found that the reduction of miR-145 was associated with EGFR abnormal activation in lung cancer cells. AG1478, an inhibitor of EGFR, may restore the expression of miR-145, indicating that EGFR activation is involved in the downregulation of miR-145 in lung cancer cells. Then, the application of STAT3, AKT and ERK1/2 inhibitors and siRNA against these signaling molecules indicated that ERK1/2 or AKT instead of STAT3 was involved in the process of miR-145 downregulation by EGFR. It was confirmed that AKT through activation of the ERK1/2 signaling molecules mediated the effect of EGFR on miR-145. Furthermore, we found that EGFR downregulated miR-145 through ERK1/2 in lung cancer cells. These findings establish EGFR and miR-145 links in lung cancer cells and therefore contribute to a better understanding of the role of EGFR in lung cancer cells, and provide clues for in-depth study of miR-145 expression and a possible direction for the further increase of miR-145 in lung cancer cells.

Objective: To investigate the relationship between EGFR activation and down-regulation of miRNA-145 in lung cancer., Methods: Normal human lung epithelia cell line (BEAS-2B), human lung adenocarcinoma cell lines with wild-type EGFR (A549 and H292) and human lung adenocarcinoma cell lines with EGFR mutation (H1975 and H1650) were chosen in this study. The levels of miRNA-145 and p-EGFR were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively, and the relationship between p-EGFR and miRNA-145 levels was analyzed. The miRNA-145 levels were determined by qRT-PCR after activating EGFR with EGF or blocking EGFR signal pathway with AG1478. In addition, ERK1/2 inhibitor U0126 was used to inhibit ERK1/2 activation and then the expression of miRNA-145 was detected., Results: The miRNA-145 levels were closely negatively related with p-EGFR in lung cancer cells (r = -0.926, P = 0.024). EGF down-regulated miRNA-145 expression, particularly in BEAS-2B cells (53.0%; t = 30.993, P = 0.001) and A549 cells (42.6%; t = 14.326, P = 0.005).The miRNA-145 was up-regulated after inhibiting p-EGFR with AG1478, and significantly enhanced by 67.5% in H1975 cells when treated with AG1478 (t = 8.269, P = 0.014). The ERK1/2 signal pathway was activated by p-EGFR. U0126 restored the miRNA-145 down-regulation induced by EGFR-activation in lung cancer cells., Conclusion: The activation of EGFR down-regulates miRNA-145 expression through ERK1/2 in lung cancer cells.

The transcription factor Ikaros was originally found to function as a key regulator of lymphocyte differentiation. In this study, we provide the first evidence that Ikaros is expressed at higher levels in ovarian cancer tissues compared with normal ovarian tissues and is significantly associated with high FIGO stage and low differentiation state in ovarian serous adenocarcinoma. To this end, we transfected IK1 (full length of Ikaros) into the SKOV3 ovarian cancer cell line and examined cell biological behaviors including proliferation, migration and invasion. We found that overexpression of IK1 inhibited cell proliferation by inducing G1 arrest, accompanied by the upregulation of P27 and P21 and downregulation of cyclin D1 and D2. On the other hand, IK1 increased the migration and invasion of ovarian cancer cells, as assessed by scratch-wound assay, transwell migration assay, and invasion assay. Overexpression of IK1 significantly increased Slug but not Snail1 expression at both mRNA and protein levels. It also downregulated and upregulated E-cadherin and MMP-2, two target genes of Slug involved in migration, respectively. Furthermore, knocking down Slug abrogated IK1-mediated increase in migration and invasion. These data suggest that Slug plays an important role in IK1-induced migration and invasion. In conclusion, we show for the first time that IK1 plays a dual role in the proliferation, migration and invasion of ovarian cancer cells, providing new insights into their metastasis.

We recently demonstrated that oridonin could induce apoptosis and senescence of colon cancer cells in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the involvement of reactive oxygen species in oridonin-induced cell death and senescence was investigated in colon adenocarcinoma-derived SW1116 cells. Oridonin increased intracellular hydrogen peroxide levels and reduced the glutathione content in a dose-dependent manner. N-acetylcysteine, a reactive oxygen species scavenger, not only blocked the oridonin-induced increase in hydrogen peroxide and glutathione depletion, but also blocked apoptosis and senescence induced by oridonin, as evidenced by the decrease in Annexin V and senescence-associated β-galactosidase- positive cells and the inhibition of oridonin-induced upregulation of p53 and p16 and downregulation of c-Myc. Moreover, exogenous catalase could inhibit the increase in hydrogen peroxide and apoptosis induced by oridonin, but not the glutathione depletion and senescence. Furthermore, thioredoxin reductase (TrxR) activity was reduced by oridonin in vitro and in cells, which may cause the increase in hydrogen peroxide. In conclusion, the increase in hydrogen peroxide and glutathione depletion account for oridonin-induced apoptosis and senescence in colorectal cancer cells, and TrxR inhibition is involved in this process. Given the importance of TrxR as a novel cancer target in colon cancer, oridonin would be a promising clinical candidate. The mechanism of oridonin-induced inhibition of TrxR warrants further investigation.

Objective: To investigate the relationship between janus kinase2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway and angiogenesis in non-small cell lung cancer (NSCLC) and explore the effects on the mRNA expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by blocking JAK2/STAT3 signaling pathway., Methods: Immunohistochemistry was used to determine the expression of P-JAK2, P-STAT3 and microvessel density (MVD) in 68 NSCLC tissues and 27 normal lung tissues. And the relationship with their clinical pathological features was analyzed. Human lung cancer A549 cells were treated with different concentrations of AG490. Cell proliferation was measured by MTT assay. Western blot was performed to detect the activation of JAK2/STAT3 signaling pathway. The mRNA expressions of VEGF and bFGF were determined by RT-PCR (reverse transcription-polymerase chain reaction). A549 cells were transfected with STAT3 siRNA. And the protein of STAT3, Phos-STAT3 (P-STAT3) and mRNA levels of VEGF and bFGF were detected., Results: The activation of JAK2/STAT3 signaling pathway was closely related to MVD in NSCLC. AG490 and STAT3 siRNA could block the JAK2/STAT3 signaling pathway and down-regulated the mRNA expressions of VEGF and bFGF in lung cancer cells., Conclusion: JAK2/STAT3 signaling pathway plays an important role in the angiogenesis of NSCLC. Blocking this pathway may inhibit the expression of angiogenic cytokines. JAK2/STAT3 signaling pathway may be a critical therapeutic target for the treatment of angiogenesis in NSCLC.

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase C delta (DeltaPKC-delta). By subcellular proteome analysis, heterogeneous nuclear ribonucleoprotein K (hnRNP K) was identified as being significantly down-regulated in NSC606985-treated leukemic NB4 cells. HnRNP K, a docking protein for DNA, RNA, and transcriptional or translational molecules, is implicated in a host of processes involving the regulation of gene expression. However, the molecular mechanisms of hnRNP K reduction and its roles during apoptosis are still not understood. In the present study, we found that, following the appearance of the DeltaPKC-delta, hnRNP K protein was significantly down-regulated in NSC606985, doxorubicin, arsenic trioxide and ultraviolet-induced apoptosis. We further provided evidence that DeltaPKC-delta mediated the down-regulation of hnRNP K protein during apoptosis: PKC-delta inhibitor could rescue the reduction of hnRNP K; hnRNP K failed to be decreased in PKC-delta-deficient apoptotic KG1a cells; conditional induction of DeltaPKC-delta in U937T cells directly down-regulated hnRNP K protein. Moreover, the proteasome inhibitor also inhibited the down-regulation of hnRNP K protein by apoptosis inducer and the conditional expression of DeltaPKC-delta. More intriguingly, the suppression of hnRNP K with siRNA transfection significantly induced apoptosis. To our knowledge, this is the first demonstration that proteolytically activated PKC-delta down-regulates hnRNP K protein in a proteasome-dependent manner, which plays an important role in apoptosis induction.

To investigate the protective effects of recombinant human tumor necrosis factor receptor II: IgG Fc fusion protein (rhu TNFR: Fc) against the lipopolysaccharide (LPS) induced intestinal damage of rats and its underlying mechanism. SD rats were randomly divided into four groups: control group, rhuTNFR: Fc group, LPS group and rhu TNFR: Fc + LPS group. Mean arterial pressure (MAP) was continuously monitored and the mortality rates were assessed. The levels of TNF-alpha and its bioactivity in the serum were assessed by ELISA and flow cytometry respectively. Pathologic changes of intestinal tissue were observed by HE staining. The rats of control and rhu TNFR: Fc group all survived with stable MAP, and the low level and bioactivity of TNF-alpha in the serum were maintained. While 83% of the rats in LPS group died by 6 h with the levels and bioactivity of TNF-alpha increasing significantly. In rhu TNFR: Fc + LPS group, the mortality rate of rats dropped to 33%. The TNF-alpha level increased compared with control group but its bioactivity decreased significantly compared with LPS group. The MPO activity and content of MDA decreased significantly. The status of pathological manifestation in the intestine was also ameliorated. These data suggest that rhu TNFR: Fc could protect rats from the acute intestine injury induced by LPS through ablating the rise in serum TNF-alpha level and bioactivity as well as anti-oxidation.

Background and Objective: Synthetic CpG oligodeoxynucleotides(CpG ODN) containing unmethylated CpG dinucleotide motifs, which mimic the effects of bacterial DNA, can stimulate the host's immune defense to reject cancer cells as "non-self" signal. This study was to evaluate the antitumor effect of CpG ODN against human neuroblastoma xenografts in nude mice depending on the innate immunity., Methods: The cytotoxicity of CpG ODN on neuroblastoma SK-N-MC cells was detected by WST-1 assay in vitro. Neuroblastoma xenografts were built subcutaneously in nude mice. When palpable tumor developed, the mice were randomized into normal saline (NS), non-CpG ODN, and CpG ODN groups (each group contained six mice), and were administered every other day for two weeks. When tumor grew to 5 cm3, the mice were killed to observe tumor morphology by histology and histochemistry., Results: CpG ODN had no cytotoxicity on SK-N-MC cells in vitro. Tumor volume was significantly smaller in CpG ODN group than in NS and non-CpG ODN groups at the end of the observation [(0.14+/-0.03) cm3 vs. (2.97+/-0.40) cm3 and (3.80+/-1.12) cm3, P<0.01]. On HE-stained sections, the tumor tissues of CpG ODN group showed intratumoral infiltration of inflammatory cells and large areas of necrosis, whereas those of controls showed less infiltration and no necrosis. By immunohistochemistry, the tumor tissues of CpG ODN group showed more natural killer (NK) cells and macrophages as compared with those of control groups., Conclusion: CpG ODN may have therapeutic effect on neuroblastoma in nude mice via mediating the activity of NK cells and macrophages.

Objectives: To block the synthesis of ryanodine receptor 2 (RyR2) in myocardial cells by RNA interference and to investigate its biological impact on ischemia-reperfusion (I/R) in rat myocardial cells., Methods: Rat myocardial cells were isolated and cultured for an I/R model in vitro. RNA interference technique was used to block the synthesis of RyR2 in myocardial cells. Changes of LDH level, apoptosis, RyR2 mRNA expression and cytosolic Ca(2+) concentration were analyzed accordingly., Results: Myocardial cells after I/R manipolation were severely injuried (LDH leakage, 125 IU/L vs 12 IU/L, P < 0.05), apoptosis (60.1% vs 5.5%, P < 0.05), significant cytosolic Ca(2+) overload (21.2 vs 7.6, P < 0.05) and remarkable mitochondrial membrane potential loss (37.2 vs 85.1, P < 0.05). However, no visible change of RyR2 was observed (20.1 vs 22.7, P > 0.05). Pre-treatment with RyR2 specified siRNA demonstrated suppressed expression of RyR2 (6.8 vs 20.1, P < 0.05), increased mitochondrial membrane potential (55.8 vs 37.2, P < 0.05), attenuated cytosolic Ca(2+) overload (8.6 vs 21.2) and cellular apoptosis (31.2% vs 60.1%, P < 0.05)., Conclusion: RyR2 gene silencing enables to protect myocardial cells from I/R injury in vitro.