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As a newly identified type of programmed cell death, cuproptosis may have an impact on cancer development, including clear cell renal cell carcinoma (ccRCC). Herein, we first noticed that the expression levels of cuproptosis regulators exhibited a tight correlation with the clinicopathological characteristics of ccRCC. The cuproptosis-sensitive sub-type (CSS), classified via consensus clustering analysis, harbored a higher overall survival rate compared to the cuproptosis-resistant sub-type (CRS), which may have resulted from the differential infiltration of immune cells. FDX1, the cuproptosis master regulator, was experimentally determined as a tumor suppressor in ccRCC cells by suppressing the cell growth and cell invasion of ACHN and OSRC-2 cells in a cuproptosis-dependent and -independent manner. The results from IHC staining also demonstrated that FDX1 expression was negatively correlated with ccRCC tumor initiation and progression. Furthermore, we identified the miR-21-5p/FDX1 axis in ccRCC and experimentally verified that miR-21-5p directly binds the 3'-UTR of FDX1 to mediate its degradation. Consequently, a miR-21-5p inhibitor suppressed the cell growth and cell invasion of ACHN and OSRC-2 cells, which could be compensated by FDX1 knockdown, reinforcing the functional linkage between miR-21-5p and FDX1 in ccRCC. Finally, we evaluated the ccRCC tumor microenvironment under the miR-21-5p/FDX1 axis and noted that this axis was strongly associated with the infiltration of immune cells such as CD4 + T cells, Treg cells, and macrophages, suggesting that this signaling axis may alter microenvironmental components to drive ccRCC progression. Overall, this study constructed the miR-21-5p/FDX1 axis in ccRCC and analyzed its potential impact on the tumor microenvironment, providing valuable insights to improve current ccRCC management.

As a dsRNA-dependent and interferon-induced protein kinase, PKR is involved in antiviral immune response and apoptosis mediated by various cytokines. In mammalian cells, PKR can also be activated in the absence of dsRNA. A PKR activator, PACT (PKR activating protein), also referred to as RAX (PKR-associated protein X) plays an important role. In recent years, with the increasing recognition of fish interferon system, PKR and PACT have been gradually revealed in fish. However, the function of fish PACT is unclear. In our previous work, we suggested that grass carp (Ctenopharyngodon idella) PACT must be involved in IRF2 and ATF4-mediated stress response pathways. In the present study, we found that the expression of C. idella PACT (CiPACT) and CiPKR were significantly up-regulated under the stimulation of LPS. It indicated that CiPACT and CiPKR may play an important role in response to LPS stimulation. In addition, the response time of CiPACT to LPS is earlier than that of CiPKR. It has also shown that overexpression of CiPACT in CIK cells can significantly enhance the level of p-eIF2α, induces apoptosis and translocation of Cip65 to nucleus from cytoplasm. To further understand the mechanism, we carried out the co-immunoprecipitation assay. It proved that the interaction of CiPACT and CiPKR made the phosphorylation of CiPKR. Overexpression of CiPACT induced the down-regulation of intracellular expression of bcl-2 and up-regulation of bax. However, in CiPKR knocked-down cells the expression of bcl-2 and bax were just the opposite. Therefore, the mechanism of fish PACT induces apoptosis and activates NF-кB is dependent on PKR., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In mammals, toll-like receptor 3 (TLR3) is capable of recognizing double-stranded RNA and then initiates transcription of IFN-β. TLR3 can activate the innate immune system by phosphorylating extracellular signal-regulated kinase 1 (ERK1) in the mitogen-activated protein kinase (MAPK) pathway. As a downstream signaling protein of ERK1, ribosomal protein S6 kinase alpha 3 (RSK2) is activated through the "classical" MAPK pathway. So RSK2 plays a critical role in response to innate immune system induced by TRL3. However, the innate immune mechanism of RSK2 remains indistinct in fish. In this study, we cloned and characterized a full length cDNA sequence of RSK2 from Ctenopharyngodon idella (named CiRSK2, MH844551). The full length cDNA of CiRSK2 is 3930 bp with a coding sequence of 2202 bp encoding a polypeptide of 734 amino acids. The expression of CiRSK2 was ubiquitous and significantly up-regulated under the stimulation of poly (I:C) in eight different tissues of C. idella and C. idella kidney cells (CIK). In addition, poly (I:C) stimulation also up-regulated the expression of CiERK1 mRNA in CIK cells and the phosphorylation of CiERK1. We also demonstrated that the activated CiERK1 interacted with CiRSK2 by CO-IP assay and immunofluorescence assay. To further investigate the relationship between CiRSK2 and CiERK1, we performed subcellular localization of CiRSK2 at different periods of CiERK1 stimulation. The result showed that CiERK1 can make CiRSK2 enter the nucleus. Subsequently, we found that CiRSK2 increased the transcriptional level of CiBCL-2 and protein level of CiBCL-2 significantly. Then cell apoptosis was inhibited to a certain extent. Overall, our results suggested that CiRSK2 plays important roles in fish innate immunity and is able to inhibit cell apoptosis by up-regulating CiBCL-2., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A water-soluble pectic polysaccharide HCA4S1 was isolated from Houttuynia cordata and purified by DEAE Cellulose and Sephacryl S-300 column. HCA4S1 with an average molecular weight of 21.7 kDa mainly consisted of rhamnose, galacturonic acid, galactose, and arabinose. By using partial acid hydrolysis, methylation analysis, and NMR spectra, the structure of this polysaccharide is found to have a backbone consisting of 1,4-linked α‑d‑GalA and 1,2,4-linked α‑l‑Rha. The latter was substituted at C-4 position by 1,4 linked, 1,6-linked β‑Galp, or Teminal linked β‑Gal. Bioactivity test showed that this polysaccharide might inhibit the proliferation of A549 lung cancer cell by inducing cell cycle arrest and apoptosis. The expression of cleaved caspase 3 and cyclinB1 was observed to be upregulated after the treatment with this polysaccharide. Collectively, these results suggest that the pectin HCA4S1 from Houttuynia cordata is of potential value in the treatment of lung cancer, though the underlying mechanisms remain to be further confirmed., (Copyright © 2018. Published by Elsevier B.V.)

Background: Ubiquitin specific peptidase 39 (USP39), an essential factor in the assembly of the mature spliceosome complex, has an aberrant expression in several cancer. However, its function and the corresponding mechanism on human osteosarcoma has not been fully explored yet., Methods: The mRNA and DNA copies of USP39 were increased in osteosarcoma cancer tissues compared with the one in human normal tissues according to datasets from the publicly available Oncomine database. A further western blot analysis also demonstrated an aberrant endogenous expression of USP39 in three different osteosarcoma cells. Then lentivirus-mediated short hairpin RNA (shRNA) was designed to silence USP39 in human osteosarcoma cell line U2OS, which is used to test the impact of USP39-silencing on cellular proliferation, colony formation, cell cycle distribution and apoptosis., Results: Knockdown of USP39 expression in U2OS cell significantly decreased cell proliferation, impaired colony formation ability. A further analysis indicated suppression of USP39 arrested cell cycle progression at G2/M phase via p21 dependent way. In addition, the results of Annexin V/7-AAD staining suggested the knockdown of USP39 could promote U2OS cell apoptosis through PARP cleavage., Conclusions: These results uncover the critical role of USP39 in regulating cancer cell mitosis and indicate USP39 is critical for osteosarcoma tumorigenesis.

Background: Triple-negative breast cancer (TNBC) has aggressive progression with poor prognosis and ineffective treatments. Selumetinib is an allosteric, ATP-noncompetitive inhibitor of MEK1/2, which has benn known as effective antineoplastic drugs for several malignant tumors. We hypothesized that Selumetinib might be potential drug for TNBC and explore the mechanism., Methods: After treated with Selumetinib, the viability and mobility of HCC1937 and MDA-MB-231 were detected by MTT, tunnel, wound-healing assay, transwell assay and FCM methods. MiR array was used to analysis the change of miRs. We predicted and verified CUL1 is the target of miR-302a using Luciferase reporter assay. We also silenced the CUL1 by siRNA, to clarify whether CUL1 take part in the cell proliferation, migration and regulated its substrate TIMP1 and TRAF2. Moreover, after transfection, the antagomir of miR-302a and CUL1 over-expressed plasmid into HCC1937 and MDA-MB-231 cell accompanied with the Selumetinib treatment, we detected the proliferation and migration again., Results: Selumetinib reduce the proliferation, migration, triggered apoptosis and G1 arrest in TNBC cell lines. In this process, the miR-302a was up-regulated and inhibited the CUL1 expression. The later negatively regulated the TIMP1 and TRAF2. As soon as we knockdown miR-302a and over-expression CUL1 in TNBC cells, the cytotoxicity of Selumetinib was reversed., Conclusions: MiR-302a targeted regulated the CUL1 expression and mediated the Selumetinib-induced cytotoxicity of triple-negative breast cancer.

The first-in-class compound MLN4924 is a small molecule inhibitor that selectively inactivates NEDD8-activating enzyme (NAE). The anticancer effects of MLN4924 have been attributed to impaired neddylation of Cullin proteins. Here, we show that treatment of T-cell acute lymphoblastic leukemia (T-ALL) cells with MLN4924 potently suppressed the neddylation of Cullins and the oncogenic growth of T-ALL cells in-vitro. Moreover, MLN4924 induced disease regression in an in vivo xenograft model. MLN4924 also induced cell cycle arrest at G2 phase and apoptosis in T-ALL cells. However, inhibition of the neddylation of Cullins alone could not explain the effects of MLN4924 in T-ALL cells. Gene expression profiling indicated ribosome function, steroid biosynthesis, and hematopoietic cell lineage pathways were affected by MLN4924 treatment. MLN4924 also induced nucleolar disruption, suggesting nucleolar stress signaling might contribute to the anticancer effects of MLN4924 in T-ALL cells. In addition, MLN4924 treatment reduced 14-3-3ξ\δ protein levels in T-ALL cells. Thus, MLN4924 may inhibit T-ALL cell proliferation via several pathways., Competing Interests: The authors declare no conflicts of interest.

Aim: Multi-drug resistance poses a critical bottleneck in chemotherapy. Given the up-regulation of mTOR pathway in many chemoresistant cancers, we examined whether sirolimus (rapamycin), a first generation mTOR inhibitor, might induce human osteosarcoma (OS) cell apoptosis and increase the sensitivity of OS cells to anticancer drugs in vitro., Methods: Human OS cell line MG63/ADM was treated with sirolimus alone or in combination with doxorubicin (ADM), gemcitabine (GEM) or methotrexate (MTX). Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry, respectively. MiRNAs in the cells were analyzed with miRNA microarray. The targets of miR-34b were determined based on TargetScan analysis and luciferase reporter assays. The expression of relevant mRNA and proteins was measured using qRT-PCR and Western blotting. MiR-34, PAK1 and ABCB1 levels in 40 tissue samples of OS patients were analyzed using qRT-PCR and in situ hybridization assays., Results: Sirolimus (1-100 nmol/L) dose-dependently suppressed the cell proliferation (IC50=23.97 nmol/L) and induced apoptosis. Sirolimus (10 nmol/L) significantly sensitized the cells to anticancer drugs, leading to decreased IC50 values of ADM, GEM and MTX (from 25.48, 621.41 and 21.72 μmol/L to 4.93, 73.92 and 6.77 μmol/L, respectively). Treatment of with sirolimus increased miR-34b levels by a factor of 7.5 in the cells. Upregulation of miR-34b also induced apoptosis and increased the sensitivity of the cells to the anticancer drugs, whereas transfection with miR-34b-AMO, an inhibitor of miR-34b, reversed the anti-proliferation effect of sirolimus. Two key regulators of cell cycle, apoptosis and multiple drug resistance, PAK1 and ABCB1, were demonstrated to be the direct targets of miR-34b. In 40 tissue samples of OS patients, significantly higher miR-34 ISH score and lower PAK5 and ABCB1 scores were detected in the chemo-sensitive group., Conclusion: Sirolimus increases the sensitivity of human OS cells to anticancer drugs in vitro by up-regulating miR-34b interacting with PAK1 and ABCB1. A low miR-34 level is an indicator of poor prognosis in OS patients.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) serves as an extracellular signal triggering apoptosis in tumor cells. However, the molecular mechanisms leading to the apoptosis are largely unknown. To characterize the molecular events involved in TRAIL-induced apoptosis, we examined the association of reactive oxygen species (ROS) in human adenocarcinoma HeLa cells. In this study, we show strong ROS accumulation upon TRAIL induction, with activation of caspases, followed by apoptosis. The pre-treatment with gamma-glutamylcysteinylglycine or estrogen, both effective antioxidants, significantly attenuated TRAIL-induced apoptosis through the reduction of ROS accumulation and diminished caspases activity. Furthermore, zVAD-fmk, an inhibitor of pan-caspase, effectively inhibited the activation of caspases and prevented apoptosis by TRAIL, although TRAIL-induced ROS generation was not attenuated. These data indicate that ROS may play a role as an upstream mediator of caspases. Taken together, our results suggest that oxidative stress mediates TRAIL-induced apoptosis in HeLa cells.

As a newly identified type of programmed cell death, cuproptosis may have an impact on cancer development, including clear cell renal cell carcinoma (ccRCC). Herein, we first noticed that the expression levels of cuproptosis regulators exhibited a tight correlation with the clinicopathological characteristics of ccRCC. The cuproptosis-sensitive sub-type (CSS), classified via consensus clustering analysis, harbored a higher overall survival rate compared to the cuproptosis-resistant sub-type (CRS), which may have resulted from the differential infiltration of immune cells. FDX1, the cuproptosis master regulator, was experimentally determined as a tumor suppressor in ccRCC cells by suppressing the cell growth and cell invasion of ACHN and OSRC-2 cells in a cuproptosis-dependent and -independent manner. The results from IHC staining also demonstrated that FDX1 expression was negatively correlated with ccRCC tumor initiation and progression. Furthermore, we identified the miR-21-5p/FDX1 axis in ccRCC and experimentally verified that miR-21-5p directly binds the 3′-UTR of FDX1 to mediate its degradation. Consequently, a miR-21-5p inhibitor suppressed the cell growth and cell invasion of ACHN and OSRC-2 cells, which could be compensated by FDX1 knockdown, reinforcing the functional linkage between miR-21-5p and FDX1 in ccRCC. Finally, we evaluated the ccRCC tumor microenvironment under the miR-21-5p/FDX1 axis and noted that this axis was strongly associated with the infiltration of immune cells such as CD4+ T cells, Treg cells, and macrophages, suggesting that this signaling axis may alter microenvironmental components to drive ccRCC progression. Overall, this study constructed the miR-21-5p/FDX1 axis in ccRCC and analyzed its potential impact on the tumor microenvironment, providing valuable insights to improve current ccRCC management. [ABSTRACT FROM AUTHOR]

Background: Dysregulated noncoding RNAs participated in progressions of cervical cancer. Purpose To verify impacts of KCNQ1OT1 on modulating progressions of cervical cancer cells. Method: Expressions of KCNQ1OT1, miR‐1270, and LOXL2 were analyzed through RT‐qPCR and protein expressions of LOXL2, p‐AKT, and AKT were validated using western blot. Bindings of miR‐1270 with KCNQ1OT1 or LOXL2 were verified using luciferase reporter assay. CCK‐8 and flow cytometry evaluated cell viability and apoptosis, respectively. The PI3K/AKT signaling pathway suppressor, LY294002, was applied to treat the cells and the changes of KCNQ1OT1 expression and LOXL2, p‐AKT, and AKT protein expressions were examined. Results: KCNQ1OT1 expression was the highest in HeLa cells but lowest in SiHa cells whose upregulation improved the viability but inhibited the apoptosis in SiHa cells while knockdown of KCNQ1OT1 caused opposite results in HeLa cells. MiR‐1270 was sponged and negatively modulated by KCNQ1OT1. MiR‐1270 mimics caused low viability and high apoptosis of SiHa cells but miR‐1270 inhibitor reverse its roles in HeLa cells. LOXL2, the target of miR‐1270, positively interplayed with KCNQ1OT1 but had negative interaction with miR‐1270. LOXL2 overexpression promoted viability and decreased apoptosis of SiHa cells but knockdown of LOXL2 restored its effects in HeLa cells. Moreover, LOXL2 and phosphorylated AKT (p‐AKT) protein expressions were downregulated by suppressed KCNQ1OT1 and LOXL2 and miR‐1270 mimics but promoted by overexpressed KCNQ1OT1 and LOXL2 and miR‐1270 inhibitor. Additionally, LY294002 treatment caused low KCNQ1OT1 RNA expression and decreased LOXL2 and p‐AKT protein expressions. Conclusion: KCNQ1OT1/miR‐1270/LOXL2 axis modulated viability and apoptosis of cervical cancer cells. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid (ATRA) has demonstrated notable success in the treatment of acute promyelocytic leukemia (APL) by inducing granulocytic differentiation. The underlying mechanisms of ATRA therapeutic effects have not been entirely clarified. Here, we reported that the regulation of neddylation, a ubiquitination-like post-translational modification, was involved in the treatment of ATRA on APL. Treating APL cells with ATRA led to the degradation of UBA3, a subunit of neddylation E1. Lysosome-autophagy pathway but not proteasome pathway was responsible for the degradation of UBA3. Neddylation suppression in APL cells was capable of inducing apoptosis, differentiation and proliferation inhibition, suggesting a pivotal role of neddylation in APL cells. ATRA treatment also led to UBA3 degradation in primary APL cells. Taken together, our findings indicated that neddylation was important to maintain the malignant features of APL cells, and suppression of neddylation was involved in the effects of ATRA on APL cells. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is the most common lethal malignancy and a leading cause of malignancy-associated death in many countries, but mainly in Asia. Expression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) protein is involved in the growth of various human cancers, including HCC. NQO1 is considered an inhibitor of cancers. The present study aimed to investigate the function and mechanism of NQO1 in HCC. In this study, we found that NQO1 overexpression decreased HCC cell SK-hep-1 and Hep3B cell proliferation and induced apoptosis. The apoptosis-associated gene Bax, Bcl-2, and caspase-3 expression was also measured, with western blot results showing that NQO1 overexpression inhibits Bcl-2 expression and promotes Bax and caspase-3 expression, whereas NQO1 silencing plays a contrasting role. In addition, NQO1 activated AMP-activated protein kinase (AMPK) and proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and the AMPK inhibitor compound C blocked NQO1-induced PGC-1α activation. Furthermore, the AMPK inhibitor compound C or PGC-1α siRNA partially abolished NQO1-induced cell apoptosis and proliferation inhibition in HCC cells. Taken together, our results demonstrate that NQO1 overexpression induces HCC cell apoptosis and proliferation inhibition through the AMPK/PGC-1α pathway. [ABSTRACT FROM AUTHOR]

Bufalin, a major component of the Chinese medicine ChanSu, which is prepared from the skin and parotid venom glands of toads, has shown cytotoxicity in several malignant tumors. Here, we reported that bufalin inhibited proliferation and induced mitochondria-dependent apoptosis in U-2OS and Saos-2 osteosarcoma cells with intracellular reactive oxygen species (ROS) production. By microRNA (miR) array analysis and quantitative reverse transcription polymerase chain reaction, we found that miR-221 was downregulated after treatment with bufalin. In accordance with TargetScan prediction and luciferase reporter assay, Bcl2 binding component 3 (BBC3) was the direct target of miR-221. Furthermore, upregulating miR-221 by its MIMIC and suppressing BBC3 by small interfering RNA (siRNA) reversed the effects of bufalin on osteosarcoma cells. Collectively, our data indicate that bufalin inhibits cell proliferation and induces mitochondria-dependent apoptosis in osteosarcoma cells through downregulating miR-221 and triggering BBC3 expression. [ABSTRACT FROM AUTHOR]